Creation of a Site-Directed Mutant of Hen Egg White Lysozyme Working Toward Site-Specific 
Oxidation as it Relates to Protein Structure  
 
 
 
by 
Eric Mensah 
 
 
 
Submitted in Partial Fulfillment of the Requirements 
For the degree of  
Masters of Science 
in the 
Chemistry 
Program 
 
 
 
 
Youngstown State University 
August, 2009 
 
 
 
 
Creation of a Site-directed Mutant of Hen Egg White Lysozyme Working Toward Site-Specific 
Oxidation as it relates to Protein Structure 
 
Eric Mensah 
 
I hereby release this thesis to the public. I understand that this thesis will be made available from 
OhioLINK ETD center and the Maag Library circulation Desk for public access. I also authorize 
the University or other individuals to make copies of this thesis as needed for scholarly research. 
 
Signature: 
 
Eric Mensah, Student                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  D a t e  
 
Approvals: 
 
 
Dr. Michael A. Serra, Thesis Advisor                                                                                                                                                                                           Date 
 
 
Dr. John A. Jackson, Committee Member                                                                                                                                                                                         D a t e  
 
 
D r .  P e t e r  N o r r i s ,  C o m m i t t e e  M e m b e r                                                                                                                                                                                                           Date 
 
 
Peter J. Kasvinsky, Dean of School of Graduate Studies   & Research                                                                  D a t e  
III 
 
Thesis Abstract 
                Metal catalyzed oxidation of protein involves a reaction between hydrogen peroxide and 
protein-bound metal ions. Production of the highly-reactive hydroxyl radical leads to oxidative 
damage in the immediate vicinity of the bound-metal ion. To examine the relationship between 
protein structure and oxidative damage a series of site-directed mutants of hen egg white lysozyme 
(HEWL) were created. This project focuses on generation of the site directed mutant where residue 
His15 will be altered to a serinyl residue (H15S), also work on the preliminary purification and 
characterization of the putative mutant would be touched on.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
IV 
 
Acknowledgements 
I would like to express my profound gratitude to my advisor, Dr. Michael Serra, for firstly 
accepting to be my advisor and whose keen interest, attention, care and driving force has made the 
timely completion of this work possible. I would also like to thank my committee members Dr. 
John Jackson and Dr. Peter Norris whose motivation can never be over emphasized.  However, I am 
extremely grateful to Dr. Mincey for allowing me into his laboratory as much as I needed to use it. 
I would also like to acknowledge and thank my family especially my wife, my mother, and 
my father for their emotional support and encouragements in all this period. I owe a lot to my 
brothers and sisters, for always calling to find out how life was treating me in a far away land, and 
also edging me on since education is the key to open life’s mystery doors. I cannot end without 
showing my strongest appreciation to my best friend and mentor Christmas Wishart who was 
always there for me when I needed psychological help. 
Finally, I realized that all these would have not come to pass without the financial support of 
the YSU School of Graduate Studies, and the esteemed faculty members of YSU Chemistry 
Department.     
 
 
 
 
 
 
V 
 
Table of Contents 
 
Title page .............................................................................................................................................. i 
Signature page ...................................................................................................................................... ii 
Abstract ............................................................................................................................................... iii 
Acknowledgements ............................................................................................................................. iv 
Table of contents .................................................................................................................................. v 
List of tables ...................................................................................................................................... viii 
List of figures ...................................................................................................................................... ix 
List of symbols and Abbreviations ..................................................................................................... xi 
Chapter I: Introduction ......................................................................................................................... 1 
Free radicals and Reactive oxygen species .......................................................................................... 2 
Anti-oxidant defense mechanism ......................................................................................................... 6 
Metal catalyzed oxidation and site-specific oxidation ......................................................................... 7 
Oxidative stress .................................................................................................................................... 8 
Protein modification ........................................................................................................................... 12 
Cross-linking ...................................................................................................................................... 12 
Peptide bond cleavage ........................................................................................................................ 13 
Oxidation of amino acid residue side chain ....................................................................................... 16 
Protein carbonylation ......................................................................................................................... 17 
Conformational changes and effects on structure and function of protein ........................................ 19 
Statement of the Research problem ................................................................................................... 19 
Chapter II: Materials and methods ..................................................................................................... 21 
Section I: Generation of the mutant gene by site-directed mutagenesis ............................................ 21 
pPICZα A cloning reaction and chemical transformation ................................................................. 21 
Analysis of plasmid for insert by PCR .............................................................................................. 22 
VI 
 
Sequencing ......................................................................................................................................... 23 
Section II: Transformation and expression of protein in Pichia pastoris .......................................... 23 
Transformation of Pichia strains ....................................................................................................... 27 
Restriction digest by Bstx................................................................................................................... 27 
Preparation of P. pastoris competent cells ........................................................................................ 27 
Transformation of P. pastoris ............................................................................................................ 27 
PCR analysis of transformants ........................................................................................................... 28 
Small-scale protein expression .......................................................................................................... 30 
Expression in BMGH/BMMH media ................................................................................................ 31 
Expression in MGYH/MMH media ................................................................................................... 32 
Analysis by Bradford assay ............................................................................................................... 32 
Analysis by SDS-PAGE .................................................................................................................... 33 
Lysozyme enzyme assay .................................................................................................................... 34 
Scale-up expression ........................................................................................................................... 35 
Section III: Purification and characterization of mutant protein ....................................................... 35 
Separation by HPLC .......................................................................................................................... 36 
Chapter III: Results ............................................................................................................................ 37 
Section I: Generation of the mutant gene, transformation of E. coli and P. pastoris ........................ 37 
Section II: Expression of protein in P. pastoris ................................................................................. 43 
Small-scale expression ....................................................................................................................... 43 
Scale up expression and purification ................................................................................................. 47 
Enzyme assay ..................................................................................................................................... 51 
Chapter IV: Discussion ...................................................................................................................... 54 
Different ways of increasing high yield secretion of the mutant protein ........................................... 58 
Conclusions ........................................................................................................................................ 58 
VII 
 
References .......................................................................................................................................... 60 
Appendix A ........................................................................................................................................ 64 
 
 
 
 
                                    
  
                         
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
VIII 
 
List of Tables 
Tables 
 
 2-1: BSA samples for standard Curve .............................................................................................. 33 
 3-1: Measurement of concentration in the various media at different time points ........................... 44 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
IX 
 
List of Figures 
Figures 
 
1-1:  Proposed reaction mechanism of dityrosine formation .......................................................... 12 
1-2:  Cleavage of the polypeptide chain by the diamide or the α-amidation pathway ................... 14 
1-3:     Oxidative cleavage of a glutamyl residue (A) and an aspartyl residue (B) ............................ 15 
1-4:     Polypeptide bon cleavage by oxidation of prolyl residues ..................................................... 16 
1-5:   Generation of carbonyl derivatives of protein:  ...................................................................... 18 
1-6:   Structure of hen egg white lysozyme highlighting the position of the only histidyl residue  21 
2-1:     5' AOX1 sequencing primer (A) and 3' AOX1 sequencing primer (B) .................................. 22 
2-2:   Yeast vector pPICZα A showing Xho I and Xba I sites ......................................................... 26 
3-1:     Agarose gel of mutant H15S .................................................................................................. 37 
3-2:   Agarose gel showing isolated plasmid of colonies EcoxA1, EcoxB2, EcoxC3 and EcoxD4 38 
3-3:     Agarose gel showing isolated plasmid of colonies EcoxE5, EcoxF6, EcoxG7, EcoxH8,  
            EcoxI9, EcoxJ10 .................................................................................................................... 38 
3-4:     1% agarose gel of PCR product of colonies EcoxA1, EcoxB2, EcoxC3 and EcoxD4  ......... 39 
3-5:   1% agarose gel of PCR product of Colonies EcoxE5, EcoxF6, EcoxG7, EcoxH8, EcoxI9 and   
          EcoxJ10 .................................................................................................................................. 40 
3-6:     Partial electropherogram of the gene sequence ...................................................................... 41 
3-7:     % agarose gel of linearized pPICZα A-H15S ........................................................................ 42 
3-8:   PCR product of the plasmid in the vector pPICZα A isolated from the yeast cell ................. 43 
3-9:     Standard curve of absorbance versus concentration BSA ...................................................... 44 
3-10:   Change in secretion amount of mutant protein with induction time ...................................... 45 
3-11:   SDS-PAGE pattern of fractions collected from BMMY ........................................................ 46 
3-12:   SDS-PAGE analysis of 24 hours and 72 hours culture .......................................................... 47 
3-13:   Graph of absorbance versus tube numbers gel filtration separation of mutant protein .......... 48 
3-14:   SDS-PAGE analysis of gel filtration fractions ....................................................................... 49 
3-15:   SDS-PAGE of dialyzed sample of 24 hour culture ................................................................ 50 
X 
 
3-16:   Chromatogram of native lysozyme eluted in phosphate buffer pH 6.0 .................................. 51 
3-17:   Graph of lytic activity of native hen egg white lysozyme  ..................................................... 52 
3-18:   Graph of lytic activity of the crude sample collected from 24 hour culture in BMMY  ........ 52 
3-19:   Graph of lytic activity of fractions collected in peak 2 from gel filtration chromatography . 53 
4-1:     1% agarose gel of Xho I digested H15S ................................................................................. 59 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
XI 
 
List of Symbols and Abbreviations 
 
ROS ............................................................................................................... Reactive Oxygen Species 
H
2
O
2
 ........................................................................................................................ Hydrogen peroxide 
LOOH ............................................................................................................................ Lipid peroxide 
RNA ........................................................................................................................ Ribose nucleic acid 
DNA DNA  ......................................................................................................  Deoxyribonucleic acid 
Cu………………………………………………………………………………………………Copper  
Fe………………………………………………………………………………………………….Iron 
M
n+
………………………………………………………………   Metal ion in the n
+
 oxidation state 
OH
-
……………………………………………………………………………………Hydroxyl anion 
M .................................................................................................................................................. Molar 
s .................................................................................................................................................. Second 
C………………………………………………………………………………………………..Carbon  
UV…………………………………………………………………………………………Ultra violet 
1
∆GO
2
 ....................................................................................................................  Singlet oxygen state 
pKa ................................................................................................. Ionization constant for a weak acid  
pH………………………………………………………………………………Hydrogen ion activity  
GSH-Px ............................................................................................................. Glutathione peroxidase 
NADPH  ........................................................................ Nicotinamide adenine dinucleotide phosphate 
MCO  ........................................................................................................... Metal catalyzed oxidation 
O
2
……………………………………………………………………………………………...Oxygen  
His……………………………………………………………………………………………Histidine 
Ser………………………………………………………………………………………………Serine 
Zn…………………………………………………………………………………………………Zinc 
SOD .................................................................................................................... Superoxide dismutase 
OS ................................................................................................................................ Oxidative stress 
Ca……………………………………………………………………………………………..Calcium 
PKC………………………………………………………………………………….Protein kinase C 
XII 
 
LDL  ................................................................................................................. Low density lipoprotein 
DNPH  ........................................................................................................ 2,4-dinitrophenylhydrazine 
cm…………………………………………………………………………………………Centimeters 
nm  ....................................................................................................................................... Nanometer 
V………………………………………………………………………………………………Voltage 
SDS  ................................................................................................................. Sodium dodecyl sulfate 
SDS-PAGE  ............................................ Sodium dodecyl sulfate-polyacrylamide gel electrophoresis 
Tris……………………………………………………………..Tris (hydroxymethyl) aminomethane  
PCR .............................................................................................................. Polymerase chain reaction  
m .................................................................................................................................................... milli  
ATP…………………………………………………………………………..Adenosine triphosphate 
µL…………………………………………………………………………………………...Microliter 
µg…………………………………………………………………………………………..Microgram 
S.O.C ...................................................................................................................... Super optimal broth 
α …………………………………………………………………………………………………alpha 
AOX  ............................................................................................................................ Alcohol oxidase 
DHAS  ........................................................................................................ Dihydroxyacetone synthase 
BSA .................................................................................................................... Bovine serum albumin 
OD  ................................................................................................................................. Optical density 
DMSO ..................................................................................................................... Dimethyl sulfoxide 
PEG  ....................................................................................................................... Polyethylene glycol 
YNB  ...................................................................................................................... Yeast nitrogen base 
DI…………………………………………………………………………………….Deionzed water 
rpm  .................................................................................................................... Revolution per minute 
HPLC  ................................................................................. High performance liquid chromatography 
TAE……………………………………………………………………………….Tris-acetate-EDTA 
AGC  ............................................................................................................................................ Serine 
CAC ........................................................................................................................................ Histidine 
kD…………………………………………………………………………………………Kilo Dalton 
XIII 
 
HEWL  ........................................................................................................... Hen egg white lysozyme 
GAP.......................................................................................................... Glyceraldehyde-3-phosphate 
µM………………………………………………………………………………………...Micromolar 
NEB..................................................................................................................... New England Biolabs 
 
1 
 
CHAPTER I: INTRODUCTION 
Reactive oxygen species (ROS) include the free radicals superoxide radical (O
2
•-
), and the 
hydroxyl radical (HO
•
), and non-radicals such as singlet oxygen, hydrogen peroxide (H
2
O
2
), and 
lipid peroxides (LOOH).  The most reactive ROS by far is the hydroxyl radical which reacts 
under diffusion controlled rates causing oxidative damage to all types of biomolecules, including 
proteins, RNA, DNA, carbohydrates, and lipids.  ROS have been implicated in many disease 
states such as premature aging, Alzheimer’s disease, amyloid beta, amyotrophic lateral sclerosis, 
Parkinson’s disease, Friedreich ataxia and Huntington’s disease.  Mutant proteins that block the 
transport of essential nuclear-encoded mitochondrial proteins to mitochondria, interact with 
mitochondrial proteins and disrupt the electron transport chain, induce free radicals, cause 
mitochondrial dysfunction, and ultimately damage neurons.
1, 2, 3 
ROS can be generated via various environmental and physiological pathways. Exogenous 
sources include ionization radiation or visible light in the presence of sensitizers, pollutants such 
as asbestos, ozone, and cigarette smoke. Endogenous sources include byproducts of aerobic 
respiration in the mitochondria, cytochrome, xenobiotics, and reactions involving transition 
metal ions and endogenously produced hydrogen peroxide.  The reactions which involve 
transition metal ions such as Cu(I) and Fe(II) and hydrogen peroxide are the most important 
source of ROS in vivo (equation 1).
4
 
                    M
n+
  +   H
2
O
2
     M 
(n+1)
   +    HO
•
 +    OH
- 
                               (1) 
Oxidation is one of the major degradation pathways for protein pharmaceuticals. 
Methionine, cysteine, histidine, tryptophan, and tyrosine are the amino acid residues most 
susceptible to oxidation due to their high reactivity with various reactive oxygen species. 
2 
 
e
‐
 + 2H
+
 
e
‐
  e
‐
e
‐
+ H
+
Superoxide 
   anion 
Hydrogen 
peroxide
Hydroxyl 
radical
Oxidation during protein processing and storage can be induced by contaminating oxidants, 
which is catalyzed by the presence of transition metal ions and induced by light.
5
  
Free Radicals and Reactive Oxygen Species 
For many years the existence of free radicals in biological systems was dismissed as 
either non-existent or simply an unimportant curiosity.  However, due to improved 
investigational techniques, this view has changed rather dramatically. A free radical is defined as 
a chemical species possessing an unpaired electron that can be considered as fragments of 
molecules and which are generally very reactive.
6
  To attain stability a free radical abstracts a 
second electron from a neighboring molecule, leading to the formation of yet another free radical 
creating a self-propagating chain reaction.
7
  Molecular oxygen is kinetically unreactive and it is 
readily reduced by an electron leading to the formation of partially reduced and reactive 
compounds collectively called reactive oxygen species.  This is illustrated in equation 2.
8
 
O
2
              O
2
•-
                  H
2
O
2
                  HO
-
  +  HO
•
                       H
2
O          (2)  
    
Several metal or metalloid ions exist in multiple oxidation states and can undergo 
electron transfer reactions that are important in biological and environmental systems. There are 
endogenous metal ions such as iron, copper and cobalt that participate in oxidation-reduction 
reactions with species including molecular oxygen, superoxide, and hydrogen peroxide. The well 
known Fenton reaction between endogenous iron and hydrogen peroxide generates the extremely 
reactive hydroxyl radical (equation 3).
5,9
 Copper in the oxidation state of +1 undergoes a similar 
reaction with hydrogen peroxide. 
                                     Fe(II) + H
2
O
2
           Fe(III) +  OH
-
  +  HO
•
            (3) 
3 
 
Fe catalyst
A two step iron-catalyzed Haber-Weiss reaction emphasized how metal ions in the higher 
oxidation state are first reduced to catalyze the reduction of hydrogen peroxide to produce the 
corresponding hydroxyl radical (equation 4).  
          O
2
•-
  +  Fe(III)  Fe(II)  +  O
2
 
          Fe(II)  +  H
2
O
2
  Fe(III)  OH
-
  +  HO
•
 
          O
2
•-
  +  H
2
O
2
  O
2
  +  OH
- 
 +  HO
•
                                          (4) 
Reactive oxygen species are unavoidable intermediates of metabolic processes of living 
organisms and they may not always be harmful. Their positive actions include, involvement in 
killing bacteria by phagocytic cells and participation in platelet aggregation.
10
 There are, 
however, suggestions that the imbalance between their synthesis and neutralization may initiate 
lipid peroxidation which is the basis of the etiology of many different illnesses.
11
 The most 
reactive of them all is the hydroxyl radical. HO
•
 reacts at diffusion controlled rates, causing 
oxidative damage to all classes of biological molecules. Almost every molecule in the cell will 
react with the electrophilic HO
•
 with second order rate constants of 10
9
 - 10
10
 M
-1 
sec
1
. Ions of 
iron, copper, cobalt, and manganese are the most physiologically studied transition elements 
involved in the production of ROS.  The kinetic data of rate constants indicate that proteins are 
major targets of HO
•
.  For  instance, the rate constant of collagen with  HO
•
  is on the order of 4 x 
10
11
 M
-1
s
-1
, DNA 8 x 10
8 
M
-1
s
-1
 and albumin 8 x 10
10
 M
-1
s
-1
. Proteins are the main target of 
oxidative damage by ROS due to the fact that they are the components of biological 
molecules.
11,12
 
Radiation in the form of ultraviolet light (UV), gamma rays, microwaves, and X-rays 
through homolytic fission of water molecules generates the hydroxyl radical.  UV can produce 
singlet oxygen. Singlet oxygen is formed when pigments are exposed to radiation in the presence 
4 
 
of oxygen.  This happens when oxygen, in its lowest energy state, absorbs energy to form the 
excited singlet oxygen states (
1
∆GO
2
).  Singlet oxygen states can also be produced in biological 
systems by reaction between H
2
O
2
 and hypochlorite resulting from phagocyte acidity, the 
interaction between light and chlorophyll, and as a consequence of the interaction between light 
and rhodopsin in the eye.  For example, patients with porphyrias are sensitive to light and 
produce excessive (
1
∆GO
2
).
4 
In the laboratory 
1
∆GO
2
 can be formed either chemically by 
reaction between H
2
O
2
 and hypochlorite or photochemically through the reaction between O
2
 
and dyes such as acridine orange or methylene blue. 
 The addition of one electron to oxygen yields the superoxide free radical anion, O
2
•-
.  The 
superoxide radical anion can be produced by exposing oxygen to ionization radiation or as part 
of normal metabolism.  For example, catabolism of xanthine to uric acid catalyzed by xanthine 
oxidase as well as other flavoproteins.  It is deliberately produced during the respiratory burst of 
phagocytic cells, forming part of the body’s defense system for the destruction of invading 
organisms.  Traces of O
2
•-
 are formed when O
2 
combines with hemoglobin and myoglobin and a 
considerable quantity of O
2
•-
 is accidentally formed in the mitochondria when oxygen is reduced 
to water.  
O
2
•-
 is a base and thus accepts protons, forming the more reactive hydroperoxyl radical 
(HO
2
•
). The pKa of this reaction is ~4.5 such that under physiological conditions only one HO
2
•
 
molecule exists for every 100 O
2
•-
 molecules.  HO
2
•
 synthesis from O
2
•-
 is pH sensitive and is 
favored by acidic conditions. Although at physiological pH the abundance of HO
2
•
 is rather low 
its level will increase when the pH decreases, for instance, following ischemia or severe acidosis 
or in close proximity to the cytosolic side of the inner mitochondrial membrane. HO
2
•
 is more 
reactive than O
2
•-
 and due to its lipophilicity, would be expected to be able to cross biological 
5 
 
membranes as effectively as H
2
O
2
.
5 
This is important, because, unlike O
2
•-
, it can initiate 
peroxidation of polyunsaturated fatty acids directly. However, HO
2
•
 is still a very poor initiator 
and there is little evidence that it can cause such damage in vivo.  The level of O
2
•-
 is mainly 
controlled by both spontaneous and enzymatic dismutation producing hydrogen peroxide 
(equation 5). 
   2O
2
•-
  +   2H
+                            
H
2
O
2
  +  O
2
                                           (5) 
Hydrogen peroxide is a non-radical ROS found in biological systems that is produced by 
several different enzymatic reactions, including those catalyzed by d-amino acid oxidase, amine 
oxidase, glycollate oxidase, ureate oxidase and superoxide dismutase.  H
2
O
2 
is also formed 
during redox cycling of compounds such as the catecholamines, from microsomal cytochrome P-
450 during the processing of fatty acids and xenobiotics, from the respiratory burst of 
phagocytes, and from mitochondrial respiration.  H
2
O
2, 
like O
2
•-
, is only a weak oxidizing agent 
and not very reactive in vivo. It will react with thiols and by so doing can inactivate enzymes that 
contain an essential thiol group, such as the glycolytic enzyme glyceraldehydes-3-phosphate 
dehydrogenase.  Unlike O
2
•-
, H
2
O
2
 can readily cross cell membranes and if it encounters Fe
2+
 or 
Cu
+
 will form the more aggressive HO
•
.
4, 14 
 
Several assays have been developed to detect and estimate HO
•
 in biological systems. 
These assays detect the products of HO
•
 oxidation such as the production of ethane from 
methional (3-methylthiopropionaldehyde) and related compounds, formation of degradation 
products from tryptophan, production of methanesulfanic acid from dimethylsulfoxide, 
production of radio-labeled CO
2
 from [carboxy-
14
C]benzoic acid, and deoxyribose assay, a 
simple "test-tube" assay for determination of rate constants for reactions of hydroxyl radicals.  
6 
 
Anti-oxidant Defense Mechanisms
 
A number of antioxidants have evolved to scavenge ROS. An antioxidant is any 
substance that when present at low concentrations compared with that of an oxidizable substrate 
significantly delays or inhibits oxidation of that substrate.  Glutathione peroxidase (GSH-Px) is 
found in human tissues such as lungs, liver, platelets, and erythrocytes.
10 
This enzyme requires 
the presence of Se and free thiol groups for catalytic activity.  It controls the speed of lipid 
peroxidation, protects cell membranes against peroxidative damage, ensures correct cell 
metabolism by taking part in the regulation of many processes such as the pentose cycle. Its main 
role is the removal of H
2
O
2
 by converting it to water.  As a result, oxidized glutathione is 
formed.  The oxidized glutathione is reduced by NADPH requiring glutathione reductase. 
Superoxide dismutase (SOD) was first isolated in 1939 from bovine erythrocytes.  It was the first 
enzyme discovered whose substrate was a free radical.
2 
Its main role is to remove O
2
•-
 by 
accelerating its conversion to H
2
O
2
. Catalases in the liver, kidney, and erythrocytes dispose of 
H
2
O
2
 by converting it to water and O
2
.  Another important enzyme that sequesters transition 
metal ions such as Fe(II) is transferrin which plays an essential role in iron metabolism. A 
glycoprotein produced in the liver, transferrin binds Fe(III) tightly but reversible. At low pH 
transferrin releases the Fe(III) in the cell’s vesicles thereby preventing an iron overload of the 
cell.
16
 Sometimes these protective mechanisms are disrupted by various pathological processes, 
and antioxidant supplements such as vitamins A, D, and E are vital to combat oxidative damage.  
Although the availability of catalytically active forms of iron and copper are very low in vivo, 
when free metal ions do become available they do not exist in the free state for long, they either 
bind to other molecules or precipitate out of solution as hydroxide or phosphate salt.
17
 Most 
amino acids are susceptible to oxidative damage due to their low oxidative potential. Methionine 
7 
 
is readily oxidized to methionine sulfide by ROS with a second order rate constant of 8.5 x 10
9 
M
-1
s
-1
.  The repair enzyme methionine sulfoxide reductase is capable of reducing the oxidized 
form back to the reduced form. This redox reaction is believed to play a role in protecting other 
functional residues from oxidative damage thus functioning as an antioxidant.  The antioxidant 
function of methionine in proteins is not limited only to proteins but functions in the reduction of 
cholesterol esters and hydroperoxides to alcohols by high density lipoproteins. 
12, 18
      
Metal Catalyzed Oxidation and Site-specific Oxidation  
The reaction between metal ions and hydrogen peroxide or O
2
 to form the hydroxyl 
radical is referred to as metal-catalyzed oxidation (MCO).  Depending upon the electron donor 
system used, the reduction of O
2
 can proceed by a two-electron mechanism yielding H
2
O
2
 
directly, or by way of two sequential one-electron transfer processes leading to the formation of 
the superoxide anion followed by its dismutation to H
2
O
2 
and O
2
. Similarly, the reduction of 
Fe(III) to form Fe(II) can occur directly or via the intermediate formation of the superoxide 
anion, which can react directly with Fe(III) given Fe(II) and O
2
.  
 
It is believed that the Fe(II) 
then binds to a metal-binding site on the protein after which the protein-iron complex reacts with 
the H
2
O
2
 to generate in situ a reactive oxygen species, which reacts with side chains of amino 
acid residues in the immediate vicinity of the metal-binding site. The MCO-catalyzed oxidation 
reaction of proteins is a site-specific process involving the interaction of H
2
O
2
 and Fe(II) or 
Cu(I) at the metal binding sites of the protein.  The site-specific nature of the reaction is 
indicated by the following facts: (a) the inactivation of enzymes by MCO systems is relatively 
insensitive to inhibition by free radical scavengers, (b)
 
only one or at most only a few amino acid 
residues in the protein can be modified when proteins are subjected to free radicals, (c) most of 
the enzymes that are highly sensitive to modification by MCO systems require metal ions for 
8 
 
activity, therefore, they must contain a metal binding-site, and (d) in the case of E. coli glutamine 
synthetase, the loss of catalytic activity correlates with loss of a single histidyl and a single 
arginyl residue per unit, both of which are situated in close proximity to one of two divalent 
metal binding sites on the enzyme.
20
 Studies have also shown two histidyl residues, His18 and 
His21, which are sensitive to oxidation in human growth hormone, are located in close proximity 
to a metal ion binding site.  Surface exposed histidyl residues which do not form part of a metal-
binding site resist MCO systems.  Site-specific oxidation has also been observed in enzyme 
systems such as Cu, Zn superoxide dismutase (Cu, Zn SOD), and in vitro, Fe(II) 
dehydrogenases, glycated insulin, and low density lipoprotein (LDL).  In fact, conversion of 
histidine especially to 2-oxo-His has been suggested as an important marker for oxidative stress 
in vivo.
21
  
Oxidative Stress 
Oxidative stress is caused by the presence of reactive oxygen species which the cell is 
unable to counterbalance.  The result is damage to one or more biomolecules including DNA, 
RNA, proteins and lipids.  Generally, ROS generation and antioxidant defense mechanisms are 
in a balance and in cases such ischemia/reperfusion the balance favors ROS production resulting 
in oxidative damage to biomolecules. Such damage includes protein carbonyl formation, 
modification of amino acid residues, covalent cross-linking, and fragmentation of the 
polypeptide chain. Oxidative stress (OS) induces cellular injury by the production of free radicals 
of high reactivity, leading to membrane lipid peroxidation and oxidation of critical thiol groups, 
causing formation of homo- and hetero-proteins bound by disulfide bridges and inhibition of 
their function.
22, 23
 De Monte, et al. accumulated evidence indicating that OS is involved in 
hepatopathies such as ischemic liver, alcoholism, steatohepatitis and pathologies leading to 
9 
 
hepatic iron accumulation among others.
23
 It is also believed that OS elevates Ca
2+
 and 
presumably activates Ca
2+ 
dependant protein kinase C (PKC), an effect causing cellular injury.
22
 
Elevated levels of protein carbonyls are found in many disease states such as rheumatoid arthritis 
and in cataractogenesis.
19
 MCO products of low density lipoproteins (LDL) have been 
implicated in atherosclerosis, a condition of chronic inflammatory response in the walls of 
arteries, in large part due to the accumulation of microphage white blood cells and promoted by 
LDLs.  The presence of iron in atherosclerotic plaques has been found and is believed to be an 
important modulator of lipid peroxidation. Accumulation of protein carbonyls have been 
implicated in aging.
19
 Relationships have been found between the content of protein carbonyls 
and the cell in human erythrocyte and in cultured fibroblast,
25
 and between the content of protein 
carbonyls and subject age in human brain and in the lens tissue of the eye.  Also, in various 
diseases, the rise in protein carbonyls has been demonstrated such as premature aging, muscular 
dystrophy, respiratory distress syndrome, amyotrophic lateral sclerosis and diabetes.
 
Furthermore, histological distribution of protein carbonyls in diseases has been determined in the 
brain tissues of patient with Alzheimer’s disease.
26
 
Since oxidative stress leads to an increase in the levels of protein carbonyls, the 
formation of protein carbonyls is used as a measure of oxidative damage in various 
physiopathological conditions. Several assays have been developed to measure protein 
carbonyls. At present protein carbonyls are generally measured spectrophotometrically using     
2,4-dinitrophenylhydrazine (DNPH), which has an absorbance maximum at 370 nm and a molar 
extinction coefficient of 22,000 M
-1
cm
-1
.  Over the past 20 years the effect of aging and various 
pathological processes on the levels of carbonyl protein extracts from tissues of laboratory 
rodents and human subjects have been studied.
27 
More recently, methods using western blots 
10 
 
have been developed to identify specific proteins showing increased levels of carbonyls. 
Chaudhuri et al. have been able to quantify the protein carbonyls on specific proteins in the 
cytosol of liver using a fluorescence-based assay which employs 2-D gel electrophoresis and 
mass spectrometry.
27
 
 Giulivi et al. proposed dityrosine as a biomarker for oxidative stress.
29
 Dityrosine 
(tyrosine-tyrosine bonding) is found in several proteins as a result of UV irradiation, γ-
irradiation, aging, and exposure to oxygen free radicals.
28 
Although carbonyl assays can be used 
as a measure of protein oxidation, it must be noted these moieties may also be introduced into 
proteins by mechanisms that do not involve the oxidation of amino acid residues.  For example, 
α,β-unsaturated alkenals produced during lipid peroxidation may react with sulfhydryl groups of 
proteins to form stable covalent thioether adducts carrying carbonyl groups.  It is noteworthy that 
aromatic amino acids in proteins are not major sites of oxidation by MCO systems, whereas 
phenylalanine, tryptophan, and tyrosine are preferred targets by radicals produced during γ-
radiolysis.  These differences in specificity result from the chemical structure of the amino acids 
with putative binding sites for metals. In the presence of oxygen, considerable peptide cleavage 
occurs, with concomitant formation of carbonyl groups. Under hypoxic or anoxic conditions, 
hydroxyl radicals produced during radiolysis leads to extensive protein-protein cross-linkage via 
dityrosine and possibly other amino acid cross-linking (e.g., disulfide bridge).  Thus, specific 
post-translational modifications of amino acids in proteins are necessary to assess cellular or 
organismal injury under different oxidative stress conditions.  It is believed that covalent 
modification of amino acids may serve as a “marking” step for protein degradation. The only 
protein modification obtained under various conditions of oxidative stress and consistent with 
simple laboratory techniques is the formation of dityrosine.
28
 Dityrosine is an unusual amino acid 
11 
 
that is distinguished by the intense 420 nm fluorescence, measurable upon excitation within 
either 312 nm (alkaline solutions) or 284 nm (acidic solutions) absorption bands.  Dityrosine 
formation begins with the generation of a tyrosyl radical, radical isomerization followed 
diradical reaction, and finally enolization with overall rate constant for the process reported as    
4 x 10
8
 M
-1
 s
-1
.
29
 Thus the initial step in the formation of dityrosine involves the formation of a 
tyrosyl radical. The reduction potential of the tyrosineO
•
/tyrosineOH couple is 0.88V
30
 indicating 
that the coupled reaction needs to be highly oxidative to be thermodynamically favorable. 
Species such as hydroxyl radical are required to fulfill this condition. Once the tyrosyl radical is 
formed, the production of dityrosine involves two monomeric molecules of tyrosine-containing 
proteins, joined by intermolecular cross-linking.  The resulting protein dimer is detectable by 
SDS-PAGE under suitable conditions. Dityrosine has also been proposed as a marker of 
organismal oxidative stress. Dityrosine concentrations were found to be 100-fold higher in LDL 
isolated from atherosclerotic lesions than normal ones.
31
 Also, humans suffering from systemic 
bacterial infections had twice the concentration of dityrosine in urine than those of healthy 
individuals.
31
 
 Methionine sulfoxide, an oxidative product of methionine is another potential marker for 
oxidative stress, and it has been detected in many disease states in vivo.
12
 
 
 
 
 
 
 
12 
 
Protein Modification    
Several lines of evidence indicate that oxidative modification of protein and the 
subsequent accumulation of oxidized proteins, which could be an early indication of oxygen 
radical-mediated tissue damage, have been found in cells during aging, oxidative stress, and in 
various pathological states including premature diseases, muscular dystrophy, rheumatoid 
arthritis, and atherosclerosis. Various protein modifications including cross-linking, peptide bond 
cleavage, oxidation of amino acid side chain, and protein carbonylation have been extensively 
studied. 
Cross-linking 
Oxidative modification of proteins can give rise to intra- or inter-protein cross-linked 
derivatives by several different mechanisms such as the formation of dityrosine.  Extensive 
dityrosine can be found in several proteins when exposed to UV radiation in the presence of 
oxygen radical (Figure 1-1). 
 
Figure 1-1:   Proposed reaction mechanism of dityrosine formation 
In the literature one can find at least five other pathways that can result in the formation of inter-
protein cross-linkages.
1
  
13 
 
• •
R
2
R
1
1. Carbon-carbon covalent linkages are formed by the direct interaction of two carbon- 
centered radicals of protein derivatives formed on oxidation of the protein backbone 
or the amino acid side-chains (equation 6). 
                        P
1
─HC─ R
1
  +  P
2
─HC─ R
2
           P
1
─HC─CH─P
2  
                              (6) 
2. Interaction between malondialdehyde and other dialdehydes with amino groups of 
lysine residues in two different protein molecules (equation 7). 
    P
1
-NH
2
  +  P
2
-NH
2
  +  CH
2
(CHO)
2
            P
1
-N=CHCH
2
CH=N-P
2
  +  H
2
O    (7) 
   
3. Interaction of the products of Michael addition (product of interaction between 4-
hydroxy-2-nonenal, HNE) with any protein of an amino group lysine residue and 
lysyl residue of a second protein molecule (equation 8). 
   P
1
-HNE-CHO  +  P
2
-NH
2
  P
1
-HNE-CH=N-P
2
  +  H
2
O         (8) 
4. Interaction of carbonyl group of products of glycation of one protein with lysyl 
residue amino of a second protein (equation 9).  
         P
1
R
1
CHO  +  P
2
-NH
2
   P
1
R
1
-CH=N-P
2
  +  H
2
O          (9) 
5. Oxidation by cysteine residue reactive species (RS) of two different proteins 
(equation 10). 
         P
1
SH  +  P
2
SH  +  RS   P
1
S-SP
2
  + reduced RS        (10) 
Peptide Bond Cleavage 
The oxidation of proteins by ROS can lead to the cleavage of peptide bonds.
32
 The alkyl 
radicals, alkoxyl radicals and alkylperoxide intermediates of protein oxidation by hydroxyl 
radical can undergo cleavage by either α-amidation or diamide pathways (Fig.1-2). 
14 
 
 
Figure 1-2:   Cleavage of the polypeptide chain by the diamide or the α-amidation pathway. 
In the α-amidation pathway, the C-terminal amino acid of the fragment derived from the 
N-terminal region of the protein will exist as an amide derivative (IV) and the N-terminal amino 
acid of the fragment derived from the C-terminal portion of the protein will exist as an α-keto-
acyl derivative (V).  In contrast, the C-terminal amino acid of the fragment derived from the N-
terminal portion of the protein via the diamide pathway will exist as the diamide derivative (II) 
and the N-terminal amino acid residue of the peptide fragment derived from the C-terminal 
region of the protein will exist as the isocyanate derivative (III).
18
 
 The oxidation of glutamyl and aspartyl residues of proteins can also lead to peptide bond 
cleavage in which the N-terminal amino acid of the C-terminal fragment will exist as the N-
pyruvyl derivative (Figure1-3).
18
 
 
15 
 
A. 
 
B. 
 
Figure 1-3:   Oxidative cleavage of a glutamyl residue (A) and an aspartyl residue (B). 
 In addition, based on findings that the number of peptide fragments formed in the 
radiolysis of proteins is approximately equal to the number of prolyl residues, Schuessler and 
Schilling (1984),
33
 proposed that oxidation of prolyl residues leads to peptide bond cleavage.  
This was subsequently confirmed by Uchida, et al. (1990),
34
 showing that oxidation of proline 
residues can lead to peptide bond cleavage by a mechanism that involves oxidation of the proline 
residue to a 2-pyrrolidone derivative (Figure 1-4 A).  It is noteworthy that acid hydrolysis of 2-
pyrrolidone yields 4-aminobutyric acid (Figure 1-4 B). Thus, the presence of 4-aminobutyric 
acid in protein hydrolysates might be a measure of cleavage by the prolyl oxidation pathway.    
16 
 
A.
 
 
B. 
2H
2
O
+
N
O
R
1
O
2-Pyrrolidone
peptide
H
2
N
O
OH
4-aminobutyric
acid
R
1
COOH
 
Figure 1-4:  Polypeptide bond cleavage by oxidation of prolyl residues (A) and acid hydrolysis   
                   of 2-Pyrrolidone (B). 
 
Oxidation of amino acid residue side chains 
All amino acid residues of peptides are susceptible to oxidation by the hydroxyl radical; 
1
 
however, the products are characterized in only a few cases. To simplify the analysis of the 
modified product formed, the side chain amino acids have been grouped as: aromatic amino 
acids, sulfur containing groups and amino acids which oxidize to give carbonyl groups. Aromatic 
amino acid residues are particularly susceptible to oxidation by ROS.  Under these conditions 
phenylalanine is converted to both mono- and dihydroxy derivatives and tyrosine is converted to 
the 3,4-dihydroxy derivative.  Tryptophan is highly sensitive to oxidation by γ-radiation, which 
leads to various hydroxy derivatives, formyl-kynurenine, and 3-hydroxy-kynurenine. It is 
17 
 
believed that tyrosine and tryptophan residues are not major targets in systems with 
physiological concentrations of transition metal ions.  Supposedly these residues are usually not 
present at metal-binding sites of proteins.  In contrast to tyrosyl and tryptophanyl residues, 
histidinyl, argininyl, and lysyl residues are highly sensitive targets for free radical induced 
oxidation in systems with physiological concentration of transition metal ions. It is assumed that 
this is due to their localization in the metal-binding sites of proteins. Oxidation of histidine 
generates the 2-oxo-histidine and its ring-ruptured products (4-hydroxy glutamate, aspartate, and 
asparagine), whereas oxidation of lysine generates carbonyl products including aminoadipic 
semialdehyde.
1,47
 Proline residues are oxidized to glutamic semialdehyde, pyroglutamic acid, 2-
pyrrolidone, and 4- and 5-hydroxy derivatives.  Though cysteine and methionine residues in 
proteins are particularly susceptible to oxidation by all forms of ROS systems, in contrast to 
other amino acid residues, the partially oxidized forms of cysteine and methionine can be 
reduced by specialized enzymatic systems. Cysteine residues of certain peroxiredoxins undergo 
reversible oxidation to sulfinic acid (Cys-SO
2
H) and the reduction reaction is catalyzed by 
sulfiredoxins.
38 
 
Protein carbonylation 
As illustrated in Figures 1-3 and 1-4, direct reaction of proteins with ROS can lead to 
formation of protein derivatives or peptide fragments possessing highly reactive carbonyl groups 
(ketones, aldehydes).  As indicated in Figure 1-5, proteins containing reactive carbonyl groups 
can also be generated by secondary reactions of primary amino groups of lysine residues of 
proteins with reducing sugars or their oxidation products (glycation/glycoxidation reactions),
18
 
and also by Michael-addition reactions of lysyl, cysteinyl, or histidinyl residues with α,β-
unsaturated aldehydes formed during the peroxidation of polyunsaturated fatty acids. Since the 
18 
 
presence of protein carbonyl derivatives in cells reflects damage induced by multiple forms of 
ROS, a number of analytical procedures have been developed to determine the protein carbonyl 
content of biological materials. 
A.  
P-Lys-NH
2
ROS
R
H
OH
n
O
R
H
OH
O
n-1
R
H
OH
NH
n
Lys-P
R
OH
O
NH Lys-P
n-1
 
 
Figure 1-5:  Generation of carbonyl derivatives of proteins: (A) by glycation/glycoxidation of   
                   lysine amino groups and (B) by reactions of α,β-unsaturated aldehydes with lysine,    
                   cysteine, and histidine residues of proteins. 
 
 
 
 
 
19 
 
Conformational Changes and Effects on Structure and Function of Proteins 
Oxidation of the side chain of amino acids can lead to unfolding and conformational 
changes in proteins which consequently affect their functions.  Studies have shown that oxidation 
of more surface exposed residues have less effect on protein structure and conformation, than 
does oxidation of more buried amino acid residues.  Solvent-accessible methionine residues are 
much more readily oxidized than the buried residues in bovine growth hormone.
35 
Oxidation of 
surface exposed methionine residues do not affect the conformation of most proteins unless the 
residues are found in a pocket. In effect oxidation of buried methionine residues affect the 
protein structure. The role of methionine oxidation in the loss of catalytic activity was studied by 
site-directed mutagenesis of subtilisin, a mutant protein with Met222 changed to Ser, Ala, or Leu 
which is resistant to oxidation by high concentration of hydrogen peroxide.
12
 This was to 
minimize the increasing inactivity of the protein due to the susceptibility of Met222 to oxidation.    
Oxidation of histidine leads to the formation of 2-oxo-histidine which is further oxidizes 
to generate the ring-opened products, such as aspartate, aspartylurea and formylasparagine. 
Therefore, histidine oxidation in effect, may change protein conformation since its oxidation 
results in loss of basicity of the side chain residue, and thus alters the net charge on the protein 
and consequently affect the isoelectric point of the protein and its hydrophobicity.  
Statement of the Research Problem 
Reactive oxygen species including the superoxide radical, hydroxyl radical, singlet 
oxygen and hydrogen peroxide, are well known cytotoxins have been implicated in the etiology 
of a wide range of diseases that affect human beings. The highly reactive hydroxyl radical is 
formed when transition metal ions including Fe
2+
 and Cu
 
in the +1 state react with hydrogen 
20 
 
peroxide. The reaction between hydrogen peroxide and the transition metal ions is referred to as 
metal- catalyzed oxidation. Some researchers argue that site-specific damage caused by metal 
catalyzed oxidation system results from a “caged” reaction in which the metal ion binds in 
pockets found along the surface of the protein.  The pocket provides a protective environment for 
the production of ROS preventing them from being scavenged by the antioxidant defense system 
in vivo. It is also known that copper binds histidine residues tightly, and; therefore, oxidative 
damage occurs frequently around histidyl residues in a protein, and it might be expected that 
oxidative damage would occur more often around histidyl residues in protein.
35
 
This argues that the primary structure may be most important in determining a proteins 
susceptibility to metal-catalyzed oxidation. This research examines whether a proteins primary 
sequence or tertiary structure is most important in site-specific oxidation. The purpose of this 
thesis was to generate a site-directed mutant of hen egg white lysozyme where residue His15 
(Figure: 1-6) was altered to a serinyl residue (H15S). The thesis will also present work on the 
expression, preliminary purification, and its characterization of the mutant protein. 
                                       
Figure 1-6:  Structure of hen egg white lysozyme highlighting the position of the only histidyl         
        residue. 
 
21 
 
CHAPTER II: MATERIALS AND METHODS 
Section I: Generation of the Mutant Gene by Site-directed Mutagenesis 
  A single site-mutant was generated by the PCR overlap extension method. This gene was 
generated and cloned into pCR
®
4-TOPO
® 
(Invitrogen) previously by Anitha Muttavarapu. The 
mutant with histidine at position 15 was changed to serine. An alteration of the 5' end allowed 
for incorporation of the gene in frame with the α-factor signal sequence found on the vector 
pPICZα A. The gene was sub-cloned into the pPICZα A, and used to transform TOP10 E. coli. 
pPICZα A
 
Cloning Reaction and Chemical Transformation 
The mutant gene was cloned into the yeast vector pPICZα A (Figure 2-1). The reaction 
mixture included 20 ng of the mutant gene (H15S) obtained from the PCR of the H15S-pCR
®
4-
TOPO
®
 construct, 40 ng (4 µL) of pPICZα A (Invitrogen), 4 µL 5x Buffer (250 mM Tris-HCl, 
pH 7.6, 50 mM MgCl
2
, 5 mM ATP, 5 mM dithiothreitol, 25% (w/v) polyethyleneglycol-8000), 1 
µL 10 mM ATP, 1µL T4 DNA ligase (New England Biolabs), and nuclease free water 
(Promega) for a total volume of 20 µL. The reaction mixture was incubated at room temperature 
for 2 hours. 2 µL of the pPICZα A cloning reaction was added to 50 µL of One Shot
®
 chemically 
competent E. coli cells in a 2 mL microcentrifuge tube. The mixture was gently mixed and then 
incubated on ice for 30 minutes. The cells were immediately heat shocked at 42 °C without 
shaking for 30 seconds and the tubes were carefully transferred back to ice. 250 µL of S.O.C 
(super optimal broth) medium at room temperature was added to each tube, capped tightly, and 
shaken horizontally at 200 rpm and 37 °C for 1 hour. 30 µL of the cells plus 20 µl of S.O.C 
medium was spread on prewarmed low salt LB plates containing 25 µg/mL of Zeocin
™ 
and 
incubated overnight at 37 °C. Ten colonies (EcoxA1, EcoxB2, EcoxC3, EcoxD4, EcoxE5, 
22 
 
EcoxF6, EcoxG7, EcoxH8, EcoxI9, and EcoxJ10) were selected and individually streaked on 
fresh low salt LB plates with Zeocin
™
 for analysis of insert. Each colony was inoculated into 2 
mL low salt LB broth with Zeocin
™
 and grown overnight at 37 °C with vigorous shaking at 250 
rpm. The plasmid was isolated from the overnight culture using a QIAprep
®
 Miniprep Kit (50). 
Analysis of plasmids for insert by PCR 
50 ng of freshly isolated plasmid was thoroughly mixed with 10 µL of GoTaq™ Green 
Master Mix (Promega), 1 µL each of the primers 10 µM 5' AOX1 and 10 µM 3' AOX1 (Figure 
2-1), and then sterile water to bring the final volume to 20 µL.  The PCR cycling program 
included an initial denaturation temperature of 95 °C for 2 minutes.  This is followed by 
denaturation at 95 °C for 1 minute, annealing at 50 °C for 30 seconds, and extension for 1 minute 
at 72 °C for 30 cycles.  The final extension was held at 72 °C for 10 minutes. The PCR products 
were analyzed on a 1% agarose gel at 85 V for 70 minutes.  The gel was soaked in ethidium 
bromide to visualize the DNA.  Samples containing the plasmid were then sequenced. 
A. 5'-GACTGGTTCCAATTGACAAGC-3' 
B. 5'-GCAAATGGCATTCTGACATCC-3' 
Figure 2-1:  5' AOX1 sequencing primer (A) and 3' AOX1 sequencing primer (B)  
Analysis of plasmids for insert by restriction digests 
To further analyze for positive transformants, 100 ng of isolated plasmid was digested 
with 2 µL of 1 mg/mL BSA, 2 µL NEB buffer # 2, 2 µL XhoI and sterile water to obtain a final 
volume of 20 µL. The reaction mixture was incubated at 37 °C for 2 hours, and then 
characterized by 1% agarose gel. Uncut plasmid was run as the control. 
 
23 
 
Sequencing  
Plasmids from colonies EcoxA1, EcoxC3, EcoxE5, EcoxF6, EcoxH8, and EcoxJ10 were 
prepared for sequencing according to the Beckman Coulter CEQ 2000 Dye Terminator Cycle 
sequencing kit, and sequencing was performed on a Beckman Coulter CEQ 2000 XL DNA 
analysis system. The reaction mixture contained 7 µL (70 ng) of DNA templates, 2 µL 
sequencing primer (1.6 µM 5' AOX1), 8 µL DTCS Quick Master Mix, and 3 µL deionized 
water. The thermal cycling program consisted of three steps as follows: 96 °C for 20 seconds,   
C.50 °C for 20 seconds, 60 °C for 4 minutes, repeated 30 times and finally held at 4 °C. The 
reaction was quenched with 4 µL stop solution (1.5 M NaOAc + 50 mM EDTA) with 1 µL of 20 
mg/ml of glycerol and the DNA precipitated with cold 95% ethanol. The pellet was then rinsed 
with cold 70% ethanol, vacuum dried, and dissolved in 40 µL sample loading buffer before 
loading into the CEQ. 
Section II: Transformation and Expression of Protein in Pichia pastoris 
Pichia pastoris is quite similar to Saccharomyces cerevisiae as far as general growth 
conditions and handling.  Pichia pastoris is an industrial methylotrophic yeast that was initially 
chosen for production of single cell proteins because of its ability to grow to very high cell 
density in simple defined media.  This fermentation technology formed the basis of a highly 
efficient expression system using the methanol-inducible AOX1 promoter and vectors that 
integrate into the P. pastoris genome.
36 
The increasing popularity of this expression system can 
be attributed to several factors, most importantly: (1) the simplicity of techniques needed for the 
molecular genetic manipulation of P. pastoris and their similarity to those of Saccharomyces 
cerevisiae, one of the most well-characterized experimental systems in modern biology; (2) the 
24 
 
ability of P. pastoris to produce foreign protein at high levels, either intracellularly or 
extracellularly; (3) the capability of performing many eukaryotic post-translational 
modifications, such glycosylation, disulfide bond formation and proteolytic processing; and (4) 
the fact that the expression system is a commercially available kit. 
P. pastoris metabolizes methanol as a source of carbon and energy.  Biochemical studies 
have shown that methanol utilization requires a novel metabolic pathway involving several 
unique enzymes. The enzyme alcohol oxidase (AOX) catalyses the first step in the methanol 
utilization pathway, the oxidation of methanol to formaldehyde and hydrogen peroxide.  AOX is 
sequestered within the peroxisome along with catalase, which degrades H
2
O
2
 to oxygen and 
water.  A portion of the formaldehyde generated by AOX leaves the peroxisome and is further 
oxidized to formate and carbon dioxide by two cytoplasmic dehydrogenases, reactions that are a 
source of energy for cells growing in methanol.  The remaining formaldehyde is assimilated to 
form cellular constituents by a cyclic pathway that starts with the condensation of formaldehyde 
with xylulose 5-monophosphate, a reaction catalyzed by a third peroxisomal enzyme 
dihydroxyacetone synthase (DHAS).  The products of this reaction, glyceraldehyde 3-phosphate 
and dihydroxyacetone, leave the peroxisome and enter the cytoplasmic pathway that regenerates 
xylulose 5-monophosphate, and for every three cycles, one net molecule of glyceraldehyde 3-
phosphate is produced. Two of the methanol pathway enzymes, DHAS and AOX, are present at 
high levels in cells grown on methanol but are not detectable in cells grown on most other carbon 
sources (glucose, glycerol, or ethanol). In cells fed with methanol in fermenter cultures, to 
ascertain growth-limiting rates, AOX levels were dramatically induced, constituting >30% of 
total soluble proteins.
36, 37
 
25 
 
 There are two genes that encode alcohol oxidase activity in P. pastoris: AOX1 and AOX2; 
AOX1 is responsible for the vast majority of alcohol oxidase activity in the cell.  Expression of 
the gene AOX1 is controlled at the level of transcription. In methanol-grown cells ~5% of 
poly(A)
+
 RNA is from AOX1; however, in cells grown on most other carbon sources,  the AOX1 
message is undetectable.
36
 The regulation of the AOX1 gene involves a two step mechanism: a 
repression/derepression mechanism plus an induction mechanism.  The expression of a foreign 
gene in P. pastoris requires three basic steps: (1) the insertion of the gene into an expression 
vector; (2) introduction of the expression vector into P. pastoris genome; and (3) examination of 
potential expression strains for the foreign gene product. A variety of P. pastoris expression 
vectors and host strains are available. All expression vectors have been designed as Escherichia 
coli/P. pastoris shuttle vectors, containing an origin of replication for plasmid maintenance in E. 
coli and markers functional in one or both organisms. Most expression vectors have an 
expression cassette composed of a 0.9 kb fragment from AOX1 composed of the 5' promoter 
sequence and a second short AOX1-derived fragment with sequences required for transcription 
termination, as well as many cloning sites for gene insertion and an antibiotic resistant gene for 
positive selection in E. coli and P. pastoris (Figure 2-2).  One set of vectors the pPICZ series are 
used for intracellular expression whereas pPICZα vectors designed for extracellular expression 
of the foreign protein. The pPICZα and the pPICZ vectors contain the sh ble gene from 
Streptoalloteichus hindustanus which codes for Zeocin
™
 resistance for selection in E. coli and P. 
pastoris.  The vector pPICZα A has selectable markers for both E. coli and P. pastoris, and is 
easier to manipulate.   
 
 
26 
 
The pPICZα A vector 
                                  
                                    
Figure 2-2: Yeast vector pPICZα A (3.6 kb) and Xho I and Xba I cloning sites. 
The possible recombinant strains that can be generated are the Mut
+
 (methanol utilization 
plus) and Mut
s
 (methanol utilization slow).  The Mut
+
 is referred to as the wild type because of 
its ability to grow on methanol as the only carbon source. Mut
s
 is due to the loss of the AOX1 
gene, which results in slow growth on methanol medium. The Mut
+
 phenotype, X-33 strain of            
P. pastoris, a wild type strain and pPICZα A has been used for this research project.  
 
 
 
27 
 
Transformation of Pichia Strains 
Restriction digest by BstX1 
To linearize plasmids for transformation into Pichia pastoris strains, 10 µg of isolated 
plasmid was digested with 2 µL BstX1 buffer, 0.2 µL of 10 mg/ml BSA, 1 µL BstX1 (Promega), 
and sterile water to bring the total volume to 20 µL. The reaction mixture was incubated at 50 °C 
for 2 hours, 5 µL aliquots of which were analyzed on a 1% agarose gel to check the digest for 
complete linearization. The enzyme was heat inactivated at 65 °C for 10 minutes after the vector 
had been completely linearized.  
Preparation of P. pastoris competent cells 
10 mL of yeast peptone dextrose medium (YPD) containing 1% Zeocin™, was 
inoculated with a single colony of Pichia strains X-33, GS115 and KM71H. Samples were 
grown overnight at 28 °C in a shaking incubator. Cells were diluted from the overnight cultures 
to an optical density (OD
600
) of 0.2 in a 10 mL sample of fresh YPD. Cells were then grown for 5 
hours at 28 °C in a shaking incubator until the OD
600
 reached 1.0. Cells were centrifuged at 2500 
rpm for 5 minutes at room temperature. The pellets were resuspended in 1 mL of solution I (1 M 
sorbitol, 10 mM bicine, 3% (v/v) ethylene glycol, 5% (v/v) DMSO, pH 8.0). The cells were 
centrifuged again, and resuspended in 1 mL of solution I as competent cells. 50 µL aliquots of 
cells were distributed into 1.5 mL microcentrifuge tubes then wrapped in several layers of paper 
towels for storage in the -80 °C freezer.  
Transformation of P. pastoris  
 For each transformation, one tube (50 µL) of competent cells was thawed at room 
temperature and 5 µg of linearized plasmid and 1 mL of solution II (40% PEG 1000, 0.2 M 
28 
 
bicine, pH 8.0) were added to each tube. The tubes were flicked vertically several times to mix 
thoroughly.  The transformation reactions were incubated in a water bath for 1 hour at 30 °C with 
mixing by flicking the tubes every 15 minutes to enhance transformation efficiency. The cells 
were then heat shocked at 42 °C in a heat block for 10 minutes.  Each tube content was then split 
into two (approximately 525 µL for each tube) and 1 mL of YPD medium was added to each. 
The cells were incubated for 1 hour at 30 °C to allow expression of Zeocin™
 
resistance. Cells 
were then centrifuged for 5 minutes at 5000 rpm at room temperature (AccuSpin™ Micro, Fisher 
Scientific). Each tube of cells were resuspended in 500 µL of solution III (0.15 M NaCl, 10 mM 
bicine, pH 8.0) and combined in one tube. The combined cells are pelleted at 5000 rpm for 5 
minutes at room temperature and resuspended in 150 µL of solution III. The entire 
transformation solutions were then platted on YPDS (1% yeast extract, 2% peptone, 2% 
dextrose, 1 M sorbitol, 2% agar) plates containing 1% Zeocin™ and grown for 3 days at 30 °C. 
Analysis of P. pastoris transformants 
Ten colonies of the Zeocin™ resistant Pichia transformants were selected on MDH 
(minimal dextrose histidine medium) and MMH (minimal methanol histidine medium) to 
confirm the Mut phenotype. Using a sterile toothpick, a single colony of one transformant was 
patched in a regular pattern on both the MDH and MMH plates with the MMH plates being the 
first to be patched.  A new toothpick was used each time for all the ten colonies. Plates were 
incubated for two days at 30 °C. To differentiate between Mut
+
 and Mut
s
, two controls, 
GS115/Mut
s
 albumin and GS115/pPICZ/lacZ Mut
+
, were also streaked as two lines on the plates. 
PCR analysis of transformants 
10 mL of YPD medium containing 100 µg/mL of Zeocin™ was inoculated with a single 
colony of the transformants from MDH/MMH plates. The cells were grown overnight in a 
29 
 
shaking incubator at 30 °C. 1.5 mL of the overnight yeast culture was spun at 13,000 rpm for 1 
minute to pellet the cells.  300 µL of acid-washed 425-600 micron glass beads, 200 µL of lysis 
buffer (2% Triton X-100, 1% SDS, 2% 100mM NaCl, 1% 10 mM Tris-HCl pH 8.0, 5% 10 mM 
EDTA), and 200 µL of PCIA (50% phenol, 48% chloroform, 2% isoamyl alcohol) were added to 
the cells to resuspend the pellets. The mixture was centrifuged for 5 minutes at 13,000 rpm 
which produced a layer of glass beads at the bottom, an opaque PCIA layer in the middle, and a 
clear aqueous layer on the top. The aqueous top layer is transferred into a new 1.5 ml 
microcentrifuge tube. 200 µL buffer TE (100 mM Tris-HCl pH 8.0, 10 mM EDTA) was added to 
the original tube, and then vortexed and centrifuged for 5 minutes at 13,000 rpm. The top 
aqueous layer was added to the previous aqueous layer. 400 µL of PCIA was added to the 
combined aqueous layer, vortexed and centrifuged for 5 minutes. The top aqueous layer was 
transferred to another tube and 400 µL of PCIA was added, vortexed, and centrifuged. This was 
repeated until the opaque layer disappeared. The supernatant was transferred into another tube 
and 40 µL of 3 M sodium acetate (pH 5.2), plus 1 mL of 100% ethanol was added. The samples 
were stored at -20 °C for 30 minutes and then centrifuged at high speed for 10 minutes. The 
supernatant was discarded and the pellet washed with 200 mL 70% ethanol and vacuum dried. 
The pellets were dissolved in 20 µL 1 x TE and 1 µL RNase. 2 µL of extracted DNA was used 
for the PCR reactions. The PCR reaction included 10 µL Green Master Mix (Promega), 50 ng of 
plasmid DNA, 1 µL 10 µM 5' AOX1, 1 µL 10 µM 3' AOX1 and nuclease-free water to adjust the 
volume to 20 µL. The thermal cycle program consisted of 95 °C melting for 2 minutes followed 
by 1 minute denaturation at 95 °C, annealing at 50 °C for 30 seconds, 1 minute extension at 72 
°C for 30 cycles, and 10 minutes final extension allowed at 72 °C completing transcription.  The 
samples were analyzed on 1% agarose gel run at 85 V for 75 minutes. 
30 
 
Small-Scale Protein Expression 
Small scale protein expression was performed in different yeast media to determine 
which one gave the best expression of protein.  
Expression in BMGY/BMMY media 
 Colonies were selected from yeast strains X-33 (PeYX1, PeYX2, PeYX3, PeYX4, 
PeYX5), GS115 (PGs1, PGs2, PGs3, PGs4, PGs5) and KM71H (PkM1, PkM2, PkM3, PkM4, 
PkM5) cultured over two days on YPDS plates containing 100 mg/mL Zeocin™.  The colonies 
were grown in 25 mL of BMGY (1% yeast extract, 2% peptone, 10% 100 mM potassium 
phosphate buffer pH 6.0, 1.34% YNB (yeast nitrogen base), 4 x 10
-5 
% biotin, 1% glycerol) in a 
250 mL baffled flasks. The cell culture was grown overnight at 28 °C in a shaking incubator at 
300 rpm until the cultures reached an OD
600
 of 2.0-6.0 at approximately 16-18 hours. The cells 
were harvested by centrifuging at 11,000 rpm for 5 minutes at 4 °C. The cell pellets were 
resuspended in 150 mL BMMY (1% yeast extract, 2% peptone, 10% 100 mM potassium 
phosphate buffer pH 6.0, 4 x 10
-5 
% biotin, 1.34% YNB, 1% methanol) medium to induced 
protein expression. 0.01% of antifoam agent (Sigma) was added to prevent frothing and to 
enhance the exchange of air. 100-150 mL of culture was placed in 1 L sterile baffled flasks, 
labeled, covered with two layers of sterile cheesecloth and grown approximately four days. 500 
µL methanol was added every 24 hours to maintain induction and 1 mL of expression culture 
was collected into 1.5 mL microcentrifuge tubes at time points of 6, 12, 24, 48, 72, 96, and 120 
hours. Samples were centrifuged at 13,000 rpm at room temperature for 1 minute. Both 
supernatant and pellets were immediately frozen on dry ice/ethanol and stored in the -80 °C 
31 
 
freezer for further analysis of secreted protein, to determine the optimum time for expression of 
the secreted protein. 
Expression in BMGH/BMMH media 
25 mL of BMGH medium (10% 100 mM potassium phosphate buffer pH 6.0, 1.34% 
YNB, 4 x 10
-5 
% biotin, 1% glycerol, 0.004% histidine) was inoculated with 50 µL of cells 
stored in 15% glycerol in a 250 mL baffled flasks. The culture was grown overnight with 
shaking at 250 rpm at 28 °C.  The cells were harvested at 11,000 rpm for 5 minutes at 4 °C when 
an OD
600
 of 2.0-6.0 was reached in approximately 16 – 18 hours. 
The cells were resuspended in 150 mL BMMH medium (10% 100 mM potassium 
phosphate, buffer pH 6.0, 1.34% YNB, 4 x 10
-5 
% biotin, and 1% methanol, 0.004% histidine) to 
induce expression.  The sample was added to a sterile 1 L baffled flask with addition of 0.01% 
antifoaming agent to prevent frothing. The culture was covered with two layers of sterile 
cheesecloth and grown four approximately four days in a shaking incubator at 300 rpm at 28 °C. 
500 µL aliquots of methanol were added after every 24 hours to maintained induction.  Aliquots 
of 1 mL of the sample were collected at time points 6, 12, 24, 48, 72, 96, and 120 hours into a      
1.5 mL microcentrifuge tube, and then centrifuged at 13,000 rpm. The supernatant was decanted 
into a clean microcentrifuge tube, and both supernatant and pellets were immediately frozen on 
dry ice/ethanol and stored at -80 °C. The supernatants were analyzed for extracellular expression 
of proteins and the pellets analyzed for intracellular expression, using the Bradford protein assay 
and SDS-PAGE. 
 
 
32 
 
Expression in MGYH/MMH media 
To express the transformed yeast strains in a non buffered medium, 25 mL of MGYH 
medium (1.34 % YNB, 1 % glycerol, 4 x10
-5 
% biotin, 0.004 % histidine) was inoculated with 
colonies of yeast transformants in 250 mL sterile baffled flasks, grown at 28 ˚C in a shaking 
incubator at 300 rpm until the OD
600
 reached 2.0-6.0, after 16 hours.  The cells were harvested 
by centrifugation at 11,000 rpm for 5 minutes at 4˚C.  The cells were resuspended in 149 mL 
MMH (minimal methanol histidine) to obtain an OD
600
 of 1.0.  The culture was then placed in a 
sterile 1 L baffled flask and antifoaming agent (0.01%) was added to avoid frothing. The cells 
were grown for four days in a shaking incubator (300 rpm). 500 µL aliquots of 100 % methanol 
were added every 24 hours to maintain induction. At the time points 6, 12, 24, 48, 72, 96, and 
120 hours, 1 mL aliquots of culture were transferred into 1.5 mL microcentrifuge tubes,  and 
then centrifuged for 1 minute at room temperature.  Both pellets and supernatant were frozen on 
dry ice/ethanol and saved in the -80 °C freezer for analysis of levels of protein expression using 
Bradford assay and SDS-PAGE. 
Analysis by Bradford assay 
 The Bradford dye-binding assay is a colorimetric assay used for measuring total protein 
concentration. It involves the binding of Coomassie Brilliant blue dye to protein in an acidic 
medium shifting the absorbance from 465 nm to 595 nm with a color change from brown to blue. 
Coomassie Bradford assay reagent (100 mg of Brilliant Blue G-250, 50 mL methanol, 100 mL of 
85% w/v phosphoric acid, DI H
2
O for total of 1 L) was used to determine the total protein 
concentration using bovine serum albumin (BSA) of a known concentration as the standard.  A 
set of protein standards were prepared using 1 mg/mL BSA in (Table 2-1). 
33 
 
Table 2-1: BSA (1mg/mL) Samples for Standard Curve 
Tube Volume of BSA (µL) Volume of DI water (µL) Coomassie reagent (mL) 
Blank 0 100 3.0 
A 10 90 3.0 
B 30 70 3.0 
C 50 50 3.0 
D 70 30 3.0 
E 100 0.0 3.0 
 
100 µL of each BSA standard was pipetted into an appropriately labeled test tube and    
3.0 mL of Coomassie reagent was added to each tube, vortexed, and incubated for 10 minutes at 
room temperature. The spectrophotometer (HP 845 UV-Visible system) was zeroed using the 
blank and the measurement taken at 595 nm.  Absorbances of all standards were measured at the 
same wavelength, and a standard curve of absorbance versus BSA concentration was prepared. 
The total protein concentrations of the supernatants obtained in all the expression yeast cultures 
were measured to quantify levels of protein expression.  
Analysis by SDS-PAGE 
 The supernatants collected at various time points in all the media were analyzed by SDS-
PAGE. The supernatants stored in the freezer were thawed to room temperature and placed on 
ice. The samples for SDS-PAGE were prepared by using 15% Tris-HCl gel (Bio-Rad 
laboratories). The gels were set in the SDS chamber and the chambers were filled with 
electrophoresis running buffer (0.25 M Tris, 1.92 M glycine, 1% SDS, pH 8.3). 10 µL of each 
sample was mixed with 10 µL of protein gel loading buffer 2x (Amresco), boiled for 4 minutes 
34 
 
and insoluble material was removed by centrifugation using a table-top centrifuge.  20 µL of 
each sample was loaded in the wells, and the gel was run at 200 V for 70 minutes.  The gel was 
then stained in Coomassie brilliant blue R-250 for 1 hour, washed with DI water five times, 
destained in a destaining solution (5% methanol, 7% acetic acid, 88% DI water), and then fixed 
in fixing solution (50% methanol, 10% acetic acid, 40% DI water) for preservation. 
Lysozyme enzyme assay  
To examine whether the lysozyme mutant protein was present in an active form in the 
supernatant a lysozyme enzyme assay was performed using Micrococcus lysodeikticus. The 
assay relies on the rate of lysis of M. lysodeikticus as suggested by Shugar.
39
 One unit of enzyme 
is equal to a decrease in turbidity of M. lysodeikticus suspension of 0.001 per minute at 450 nm 
and 25 °C. The M. lysodeikticus cell suspension was prepared by adding 0.3 mg/mL of cells to 
0.1 M potassium phosphate buffer (pH 7.0). The suspension was allowed to incubate for 1 hour 
at room temperature. The assay was performed on a HP 845 UV-Visible spectrophotometer at a 
wavelength of 450 nm. The sample cell was held at 25 °C using a circulating water bath.  2.9 mL 
of suspended M. lysodeikticus was pipetted into a disposable cuvette and incubated for 4 minutes 
to equilibrate the cells.  Blank readings were obtained using the equilibrated sample. 100 µL of 
lysozyme standard in various concentrations was added and the absorbance recorded every 10 
seconds for 4 minutes.  The native lysozyme enzyme starting at 10 mg/mL was diluted several 
times until the corrected rate of 0.02 ∆A
450
/minute was obtained.  The corrected rate was 
calculated using zero order kinetics from 40-240 seconds.  The supernatant samples were 
assayed alongside the standards to avoid variation in cell substrate. 
 
 
35 
 
Scale-up of Expression    
The scale-up expression was begun once the optimization using small-scale expression 
had been achieved. Colonies which did express the protein were grown in large quantities in the 
respective media for further studies.  25 mL of BMGY was inoculated with 50 µL of the highest 
expresser yeast cells (Gps1) saved in 15% glycerol in a 250 mL baffled flask. The cell culture 
was incubated in a shaking incubator (250 rpm) at 28 °C for 16 hours until the culture reached an 
of OD
600
 of 2.0-6.0. The cells were harvested with sterile centrifuge bottles at 4 °C and 11,000 
rpm for 3 minutes. The supernatant was decanted and the cell pellet resuspended in BMMY to an 
OD
600
 of 1.0. The culture was then aliquoted into 300 mL volumes into several 2 L baffled 
flasks, covered with two layers of cheesecloth and returned to the incubator to continue to grow 
till the time of induction was reached (24 hours).  The sample was centrifuged at 13,000 rpm at 4 
°C for 10 minutes and the pellets were discarded. Supernatant was concentrated from 600 mL to 
4 mL, for purification experiments.      
Section III: Purification and Characterization of Mutant Protein 
 Preliminary purification with gel filtration chromatography was carried out to purify the 
mutant protein. 100 g of Sephacryl™ S-100 was soaked in 200 mL of water overnight to get rid 
of all preserving solution (20 % ethanol).  The water was drained by decanting.  200 mL of 
buffer (20 mM potassium phosphate, 0.4 M NaCl, pH 6.0) was added to the gel beads to form a 
thick slurry.  The gel was packed in a 90 cm x 1.5 cm column to a height of ~60 cm.  Two 
column volumes of buffer were allowed to run through it to equilibrate the stationary phase. A 2 
mL concentrated sample of the mutant lysozyme protein (0.54 mg/mL) was applied onto the 
column bed. The protein was allowed to elute with the same buffer of 20 mM plus 0.4 M NaCl, 
36 
 
pH 7.0 at 75 drops per test tube, and the absorbance of each tube was determined at 280 nm and 
a graph of A
280
 versus test tube number was plotted.  Fractions were pooled under peaks with the 
highest absorption and further concentrated using Amicon concentrators (MWCO 3000 Da). The 
fractions are dialyzed to remove low molecular weight proteins and salts and then concentrated 
to a desired volume (2 mL-4 mL) using Amicon concentrators. 
Separation by HPLC 
 HPLC samples were prepared by obtaining aliquots of protein samples dialyzed against 
20 mM potassium phosphate buffer and 0.4 M NaCl, centrifuged at high speed and the 
supernatant decanted into a clean microcentrifuge tube. The precipitate formed at centrifugation 
was dissolved in HPLC buffer A (20 mM potassium phosphate, pH 6.0), centrifuged and the 
supernatant added to the previous one.  The HPLC column was flushed with 2 column volumes 
of the high salt buffer, buffer B (20 mM potassium phosphate, 0.4 M sodium chloride, pH 6.0), 
and equilibrated for 15 minutes with the low salt buffer, buffer A in an isocratic mode.  The 
column was then equilibrated with high salt buffer B and low salt buffer A at a gradient of 10% 
for 15 minutes at a flow rate of 1.00 mL/min. 500 µL of native lysozyme at 4 mg/mL was first 
loaded onto the cation exchange column (polyCAT A™).  The protein was eluted in 40 minutes 
with the linear gradient to 100% B, and the data collected from 0-40 minutes.  Native lysozyme 
samples were run before any of the mutant protein was injected.  Samples were injected every 75 
minutes.  Various concentrations of salt in buffer B was employed to ensure complete elution of 
the mutant protein, since its elution time was lower than the anticipated time. 
Samples obtained from the size exclusion chromatography were analyzed for protein 
detection, quantification, purity, and activity.                                                                                                           
37 
 
CHAPTER III: RESULTS 
Section: I Generation of the Mutant Gene, Transformation of E. coli and P. pastoris 
The mutant gene (H15S) was isolated from pCR
®
4-TOPO® and was purified and 
amplified and used as the lysozyme mutant gene DNA template. Figure 3-1 illustrates the 
digested gene on 1% agarose gel showing a band of the right size (400 bp).  The gene was then 
sub-cloned into the Pichia vector pPICZα A, followed by transformation into competent One 
Shot
® 
TOP10 E. coli cells. The plasmid was isolated and 10 µL of sample obtained from the 
plasmid purification was analyzed by 1% agarose gel electrophoresis in 1 X TAE buffer. The gel 
was run at 85 V for 75 minutes and then soaked in ethidium bromide in 1 X TAE buffer for 15 
minutes and analyzed by the Stratagene imaging system EagleSight™ (Figures 3-2 and 3-3). 
                                                   
 
Figure 3-1:   1% agarose gel of Xho I digested mutant H15S cloned into pCR
®
4-TOPO®.  
Lane 1, 100 bp ladder; Lanes 3 and 4, and 5 H15S mutant gene from three 
different colonies.   
                                                                                  
38 
 
                                       
 
Figure 3-2:   1% agarose gel showing the isolated pPICZα A, H15S plasmid clones. Lane 1, 1 
kb ladder; Lane 3, EcoxA1; Lane 4, EcoxB2; Lane 6, EcoxC3; and Lane 7, 
EcoxD4. 
  
  
                     
Figure 3-3:   1% agarose gel showing isolated pPICZα A, H15S plasmid clones. Lane 1, 1 kb  
ladder; Lane 2, EcoxE5; Lane 3, EcoxF6; Lane 4, EcoxG7, Lane 5, EcoxH8; Lane 
6, EcoxI9, and Lane 7, EcoxJ10.   
 
39 
 
 Plasmids isolated from colonies EcoxA1, EcoxC2, EcoxE5, EcoxF6, EcoxH8, and 
EcoxJ10 were analyzed for insertion of the gene using PCR.  The PCR product was run on 1% 
agarose gel electrophoresis at 85 V for 75 minutes and stained with ethidium bromide in 1 x 
TAE buffer.  The PCR product of the plasmid included nucleotides of the pPICZα A from the 5' 
AOX1 and 3' AOX1 primers and the inserted gene.  Figures 3-4 and 3-5 illustrate PCR product 
run on 1% agarose gel showing the band of the right size (1000 bp).  
  
                       
Figure 3-4: 1% agarose gel of the PCR products using pPICZα A plus the H15S gene as  
 template. Lane 1, 1 kb ladder; Lane 2, EcoxA1; Lane 3, EcoxB2; Lane 4, EcoxC3;  
                   Lane 5, EcoxD4; and Lane 6, 100 bp ladder. 
 
       
40 
 
                      
Figure 3-5:   1% agarose gel showing the PCR products of plasmids isolated from colonies 5 to 
10. Lane 1, 1 kb ladder; Lane 2, EcoxE5; Lane 3, EcoxF6; Lane 4, EcoxG7; Lane 
5, EcoxH8; Lane 6, EcosI9; Lane 7, EcoxJ10. 
 Colonies EcoxA1, EcoxC3, EcoxE5, EcoxF6, EcoxH8 and EcoxJ10 were sequenced 
before transforming into Pichia to confirm that the gene was in frame with the α-factor signal 
sequence, using the sequencing primers 5' AOX1 and 3' AOX1. The primers are designed to 
amplify the gene along with the sequences coding for the gene’s native sequencing signal that is 
the α-factor signal of the vector. The electropherogram data obtained from plasmids isolated 
from transformed colonies, indicated that the gene had been incorporated in frame with the α-
factor signal sequence, and the mutation at position 15 points to the fact that alteration of 
histidine 15 (codon CAC) to serine 15 (codon AGC) had occurred (Figure 3-6).   
  
41 
 
 
Figure 3-6: Partial electropherogram of the gene sequence with codons AGC encodes for  
 serine. 
 
10 µg of pPICZα A-H15S was linearized by BstXI (Figure 3-7) which was then integrated 
into the Pichia pastoris genome by the EasyComp™ transformation method.  Ten colonies were 
selected after the transformation of the yeast cell. This was followed by the isolation of the 
genomic DNA from the cells, which was subsequently analyzed by PCR.  The PCR analysis 
revealed a band of 1000 bp of one of the colonies (GPs1) indicating a successful integration of 
the cloned plasmid into the yeast DNA (Figure 3-8).  
 
 
  
42 
 
                   
Figure 3-7:   1% agarose gel of linearized pPICZα A-H15S. Lane 1, 1 kb ladder; Lane 3, 
plasmid isolated from EcoxA1; Lane 4, plasmid isolated from EcoxD4, Lane 6, 
EcoxE5; and Lane 7 EcoxJ10.  
Plasmids from colonies EcoxA1 and EcoxD4 digested with BstX1 produced an intense 
band at 4000 bp. The digested DNA from these two was used to transform competent P. pastoris 
strains X-33, KM71H, and GS115. The positive integrants selected on YPDS plates were 
analyzed for the integrated gene and subsequent expression of the mutant protein.  Figure 3-8 is a 
1% agarose gel showing the PCR product of the yeast genomic DNA with a band size of ~1000 
bp.  Colony GPs1 of the GS115 strain was successfully transformed and that was used to begin 
the small-scale expression of protein. 
   
 
43 
 
 
Figure 3-8:   PCR product of the isolated yeast genomic DNA. Lane 1, 1 kb ladder; Lane 2, 
transformed GS115 strain, (GPs1). 
Section II: Expression of Protein in P. pastoris 
Small-scale expression 
 GPs1 was grown in three different media to determine the optimal conditions to express 
the mutant protein.  The secretion of the mutant protein in shake-flask cultures of the media was 
investigated by the Bradford protein assay and SDS-PAGE.  The secreted protein concentrations 
(mg/mL) in the various media were estimated from the standard curve of absorbance versus 
concentration of BSA in µg (Figure 3-9).   
44 
 
 
 Figure 3-9:  Standard curve of absorbance at 595 nm versus concentration in µg of BSA.  
The concentration of the supernatant fractions collected every 24 hours was estimated 
(Table 3-1) to determine the amount of secreted protein at the various time points.  This was 
necessary to optimize the expression of the mutant protein in large shake-flask cultures. 
 
 
Table 3-1:   Measurements of concentrations in the various media at different time points  
BMGY/BMMY 
Time (hours) 24 48 72 96 120 
Abs at 595 nm 0.3573 0.3375 0.3866 0.3986 0.3709 
Protein conc.(mg/mL) 0.199 0.1856 0.2177 0.2255 0.2074 
BMGH/BMMH 
Time/hours 24 48 72 96 120 
Abs at 595 nm 0.0141 0.0052 0.0827 0.0691 0.0571 
Protein conc.(mg/mL) 0.0000 0.0000 0.0191 0.010 0.002 
MGYH/MMH 
Time/hours 24 48 72 95 120 
Abs at 595 nm 0.1014 0.1227 0.1105 0.1103 0.1100 
Protein conc.(mg/mL) 0.0313 0.0452 0.0374 0.0371 0.0369 
45 
 
As shown in Table 3-1 the amount of mutant protein secreted in the media BMMY and 
MMH increased steadily with induction time, but shows higher concentrations at each time point 
for BMMY (Figure 3-10).  Secretion levels were at maximum at 72 hours when BMMH was 
used as the growth media and then decreased.  BMMY was selected as the medium for protein 
expression, and the induction time was pegged between 24 hours to 72 hours. 
 
                
Figure 3-10:   Change in concentration of secreted protein with induction time. 
The variations of the protein band of the mutated lysozyme of samples collected at 
various time points in BMMY are shown Figure 3-11. The band density represents secretion 
level. The result indicated a protein band that co-migrated with the native lysozyme (14.4 kD) 
for the 24 hour and 72 hour cultures. Based on the results obtained from SDS-PAGE analysis, 
‐0.05
0
0.05
0.1
0.15
0.2
0.25
0 50 100 150
Secret
i
on l
e
vel
 
(m
g/
m
l
)
Inductione time (hrs)
BMMY
BMMH
MMH
46 
 
methanol induction time for shake-flask cultures of the mutant lysozyme was set at 24 hours and 
72 hours in BMMY.  
  
 
Figure 3-11:  SDS-PAGE of fractions collected from BMMY over time: Lane 1, Precise™   
Protein Molecular Weight; Lane 2, Lysozyme Standard; Lane 4, 24 hr; Lane 5, 48 
hr; Lane  6, 72 hr; Lane 7, 96 hr; and Lane 9 120 hr. 
  
Partially concentrated and partially purified supernatant obtained from time points 24 
hours and 72 hours with 1% methanol induction was analyzed by SDS-PAGE (Figure 3-12). The 
results obtained in the SDS-PAGE gel indicated a strong and intense band close to the 14.4 kD 
band of native lysozyme was for the sample taken from the 24 hour culture as compared to 72 
47 
 
hour culture.  As a result of this, the methanol incubation time was set at 24 hours for scale-up 
expression of protein. 
 
Figure 3-12:  SDS-PAGE analysis of 24 and 72 hour cultures. Lane 2, molecular weight  
marker; Lane 4, hen egg white lysozyme; Lane 6, 24 hour supernatant; and Lane 7, 
72 hour supernatant. 
 
Scale-up expression and purification 
 The BMGY/BMMY growth culture gave the most promising results in small-scale 
expression experiments. The amount of protein secreted after 24 hours was about 0.24 mg/mL 
with 1% methanol induction. One liter batch cultures were grown. The supernatant collected was 
concentrated using the Easy Load Master Flex
®
 equipped with Minimate™ TFF Capsule down 
to about 30 mL. This volume was further concentrated to between 2-4 mL using Amicon Ultra-4 
(Ultracel-3k) concentrators. A Bradford assay of the concentrated sample gave a concentration of 
48 
 
0.43 mg/mL protein. Two milliliters of the concentrated protein was loaded onto the size 
exclusion column and eluted with a low salt phosphate buffer.  
 
Figure 3-13: Graph of absorbance versus tube number for eluted fractions separated by; using 
Sephacryl™ S-100. 
The adsorbed proteins were eluted with the starting buffer containing 0.17 M NaCl. 2 mL 
fractions were collected in each test tube. Figure 3-13 shows a plot of the absorbance at 280 nm 
versus tube numbers for fractions eluted from the column.  Fractions in the peaks were pooled, 
concentrated and analyzed by SDS-PAGE (Figure 3-14). 
    
49 
 
Figure 3-14:  SDS-PAGE analysis of gel filtration fractions: Lane 1; protein molecular weight     
marker, Lane 3; hen egg white lysozyme standard, Lane 5, fractions in peak 1, 
Lane 6; fractions in peak 2; and Lane 7, fractions in peak 3. 
 As shown in lane 5 (Figure 3-14), larger molecules were observed in fractions collected 
under peak 1 while smaller molecules are found  for fractions collected under peaks 2 and 3. To 
further purify the samples for HPLC analysis, fractions collected under peaks 2 and 3 were 
dialyzed against 20 mM potassium phosphate buffer. Precipitate formed during dialysis but was 
dissolved in a low salt buffer and preserved for further analysis. The supernatant and the 
dissolved precipitate were concentrated further down using Amicon Ultra-4 concentrators. The 
resultant concentrated protein solutions were washed with water to remove salts. The SDS-
PAGE indicated that the precipitate that formed upon dialysis did not contain a protein with a 
molecule near that of lysozyme (Figure 3-15).  
50 
 
 
Figure 3-15:  SDS-PAGE of dialyzed sample from 24 hr culture in BMMY. Lane 1, molecular  
weight marker; Lane 2, hen egg white lysozyme; Lane 4, crude sample; Lane 5 
supernatant from dialysis; Lanes, 6, 7 and 8 precipitated molecular species 
dissolved in 20 mM potassium phosphate buffer 
 
 
In attempts to separate the mutant protein from other molecular species, 500 µL aliquots 
of the crude sample, the concentrated samples from dialysis, and samples from the eluent of size 
exclusion chromatography were loaded onto a PolyCAT A™ column. The proteins were eluted 
as described in Materials and Methods. Unlike the native hen egg lysozyme, the proteins from 
cultured samples eluted from the column within 2 to 10 minutes and there were no representation 
peaks near the one for native lysozyme which eluted between 34-36 minutes (Figure 3-16) 
51 
 
 
Figure 3-16:  Chromatograms of native lysozyme (red line) and mutant protein (blue line)  
 eluted with high salt phosphate buffer pH 6.0 at a flow rate of 1.00 mL/min.                
Enzyme Assay 
 Enzyme assays were performed to determine the lytic activity of crude extracts, 
concentrated samples, dialyzed and partially purified samples of the mutant protein.  The 
absorbance at 450 nm was measured every 10 seconds for a total of 4 minutes.  The 
∆A
450
/minute was obtained using zero order kinetics fitting data from 40-240 seconds.  The lytic 
activity obtained from a 100 µL aliquot of 10 µg/mL of hen egg white lysozyme was used as a 
control (Figure 3-17).  
52 
 
                        
Figure 3-17: Graph of lytic activity of native hen egg white lysozyme. 
Lytic activity could not be detected with 100 µL aliquots of samples collected at 48 
hours, 72 hours, 96 hours and 120 hours. The 24 hour culture showed weak lytic activity when 
supernatant obtained from BMMY cultures were applied to M. lysodeikticus (Figure 3-18). 
                                 
Figure 3-18:  Graph of lytic activity of the crude sample from collected 24 hour culture in    
                    BMMY. 
53 
 
 The fractions obtained from gel filtration chromatography were analyzed for enzyme 
activity and most failed to exhibit any lytic activity, but the concentrated fraction of peak 2, did 
show detectable lytic activity (Figure 3-19). 
 
                     
Figure 3-19:  Graph of lytic activity of fractions collected in peak 2 from gel filtration  
 chromatography. 
 
 
 
 
 
 
 
 
54 
 
CHAPTER IV: DISCUSSION 
The perception that metal-catalyzed oxidation is a “caged” reaction has been discussed by 
many researchers.
46
 The “caged” system of the reaction occurs when metal ions bind in a 
protective pocket on the protein’s surface providing protection from anti-oxidants for the 
production of ROS. Other lines of evidence also suggest the availability of a metal-binding 
residue alone is sufficient to initiate metal catalyzed oxidation. To examine the importance of 
primary as opposed to the tertiary structure in site-specific oxidation, a single mutant of hen egg 
white lysozyme (HEWL)was generated. In the amino acid sequence of native HEWL (appendix 
A) His15 is found in a protective pocket (Figure 1-6), which provides protection for the 
production of ROS, and it becomes difficult for the antioxidant defense systems to scavenge. 
 HEWL was chosen as the model protein for several reasons. The enzyme is relatively 
small consisting of only 129 amino acid residues.  It is a well characterized structure and is one 
of the most extensively studied proteins having been used as a model protein in various fields of 
protein research.
40
 Several successful attempts have been made to express other forms of 
lysozyme in E. coli and P. pastoris. The unstable H5-lysozyme mutant protein with its 
hydrophobic pentapeptide (Phe-Phe-Val-Ala-Pro) at C or N-terminus has been expressed in P. 
pastoris with methanol induction. The amount of the protein secreted was 422-fold greater than 
that observed in Saccharomyces cerevisiae.
41
 Mutant N
15
 enriched HEWL has also been 
expressed in the P. pastoris system.
42, 43 
HEWL has been expressed in E. coli, however, the 
expression of active enzyme in E. coli consequently led to bacterial cell lysis due to hydrolysis of 
the peptidoglycan.
41, 44
 
In this research study, a single site mutant of HEWL has been generated. Serine was 
deemed a safe substitution for histidine based on an algorithm developed by Bordo and Argos.
40
  
55 
 
The initial steps of the experiment involved generating the single mutant H15S gene and cloning 
the gene into the yeast vector pPICZα A. The Pichia vector cloned with the H15S was used to 
transform the TOP10 strain of E. coli for maintenance of the plasmid. The insertion of the gene 
into the E. coli cells was verified by isolating the plasmid from 1.5 mL of an overnight culture of 
the cells as described in Materials and Methods. The plasmid DNA was analyzed by agarose gel 
electrophoresis and DNA sequencing.  The cloned vector was linearized and used to transform 
the Pichia strains X-33, GS115 and KM71H for the expression of the gene.  
HEWL has a molecular weight of 14.4 kD;  therefore, a protein band of ~14 kD was 
expected in the SDS-PAGE gels run with samples obtained from the various time points of 0, 24, 
48, 72, 96, and 120 hours in the different media used in the expressing the mutant protein. A 
band of the correct size was observed when the complex buffered medium BMMY was used in 
the small-scale expression with culture volume of 100-150 mL.  There was no visible band near 
14.4 kD in the SDS-PAGE gels when samples collected from media BMMH and MMH were 
analyzed.  The amount of protein secreted into the medium increased from zero hours and then 
decreased substantially after 24 hours.  Lytic activity was not detected in samples collected from 
time points 48, 72, 96, and 120 hours. There was slight detection of lytic activity with the crude 
and the purified sample of the 24 hour time point (Figures 3-18 and 3-19).  The lytic activity 
obtained for the 24 hour culture supernatant (Figure 3-18) showed greater variability as 
compared to the purified concentrated sample from gel filtration chromatography obtained for 
the same 24 hour period. 
Large scale expression in BMMY medium did not produce as much protein as expected 
in the 24 hour culture, however, an increase of about 7.5% in terms of milligram protein was 
observed compared to the amount of protein obtained in the small scale expression.  Samples 
56 
 
obtained from gel filtration were analyzed by cation exchange chromatography in an effort to 
separate the secreted protein from other molecular species however, there were no visible peaks 
that eluted close to the elution range of the native lysozyme. 
Due to low levels of protein expression some parameters such as methanol concentration 
and incubator shaking speed were subjected to changes. The amount of methanol was increased 
from 1% to 2% and the rotor speed was adjusted to 350 rpm. Nevertheless, these changes did not 
improve the yields in protein expression.  By comparison, it has been reported in the literature 
that 
15
N-labeled lysozyme expression in P. pastoris reached 20 mg/mL in five days with 0.5% 
methanol induction.
42
 It has also been reported that expression of H5-lysozyme in P. pastoris 
was 2.1 mg/mL after four days in BMMY medium.            
    In order to ensure that lower levels of secretion of the mutant protein were not due to 
intracellular protein accumulation, SDS-PAGE analysis was performed on cell lysates after 
various times of induction. In all the instances no visible band of ~14.4 kD was observed. Based 
on the low level of expression the vector cloned into the bacteria cell was cross checked by 
performing a Xho I digest (Figure 4-1).  
 
  
57 
 
               
Figure 4-1:   1% agarose gel of Xho I digest of H15S, pPICZα A plasmid. Lane 1, 1 kb ladder;  
                   Lane 3 to Lane 6, plasmids isolated from JM110 E. coli strain. 
         
The cloned plasmid used in our expression of the H15S HEWL evidently 
underwent a recombination event in the JM110 E. coli strain.  This was evidenced by 
the size of the digested band at approximately 5500 bp.  The correct band size is 
4000 bp.  This is a likely possibility for the low level of protein expression that has 
been experienced even in large cultures. Based on these results, fresh gene was 
acquired, re-cloned into fresh pPICZα A and the plasmid DNA used to transform 
fresh competent TOP10 E. coli to continue with the expression as discussed in 
chapter III.  
 
 
58 
 
Different ways of increasing high yield secretion of the mutant protein 
 The chromatographic and electrophoretic data clearly indicated that the 
mutant protein was secreted extracellularly, but expression levels were relatively low 
therefore, better expression conditions are necessary to express the mutant protein in 
high yields. There are several options that exist to improve expression levels such as 
generating strains containing multiple integrants of the vector or creating multiple 
copies of the recombinant gene integrated into the yeast genome.
31
  Another 
alternative is to introduce the P. pastoris glyceradehyde-3-phosphate (GAP) 
promoter into the expression vector instead of the AOX1 promoter.  The GAP 
promoter has been reported to secrete the recombinant esterase into the external 
medium to a concentration of 7 mg/mL.
45 
The advantage of using GAP is that 
methanol is not required for induction, nor is it necessary to shift cultures from one 
carbon source to another, making strain growth more straightforward. 
 One of the reasons for the low level of protein expression in large scale 
cultures can be attributed to relatively low bio-mass accumulation in shake-flask 
cultures (OD
600
 2.5-3.0). P. pastoris yeast thrives well in fermenter cultures with 
good bio-mass accumulation; therefore, using fermentors may also increase protein 
expression. 
Conclusions:  
 In this study the mutant lysozyme (H15S) was expressed using the P. pastoris 
expression system.  The PCR and sequence analysis results indicated that the H15S 
mutant gene was successfully cloned into the P. pastoris genome.  The analysis of 
supernatant from shake-flask cultures on SDS-PAGE revealed a band that migrated 
59 
 
with the native lysozyme (control), and was assumed to be the mutant lysozyme. 
Fractions obtained in the elution peak T2 from gel filtration were prepared and 
applied to SDS-PAGE. The SDS-PAGE investigation also revealed two protein 
bands near the migration position of the native lysozyme.  Expression of the H15S 
gene in various media was carried out according to standard protocols. Buffered 
complex media with methanol was found to be suitable for the expression of the 
protein. Expression appeared to be maximal with 1% methanol induction at 24 hour 
time point and a shaker speed of 300 rpm. However, since the secretion level of the 
mutant protein was too low (< 1mg/mL) to carry out metal-catalyzed oxidation 
studies, it would be prudent to find alternative purification techniques or different 
routes to enhance expression using P. pastoris. For example (a) additional studies 
aimed at optimizing culture conditions for the GS115 strain which has a mutation in 
the histidinol dehydrogenase gene (His4) and the X-33 strain which is the wild-type 
with Mut
+
 phenotype in which the integrated gene will be found upstream of the 5' 
AOX1 region. (b) generating a strain with multiple integrated copies of the 
expression cassette. A strain that contains multiple integrated copies of the 
expression cassette can sometimes yield more heterologous protein than single-copy 
strains.
31
   
 
 
 
 
60 
 
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64 
 
Appendix A 
                       Hen Egg White Lysozyme Gene and Amino Acid Sequence 
1-10  AAA GTC TTT GGA CGA TGT GAG CTA GCA GCG 
  Lys  Val  Phe  Gly  Arg Cys Glu Leu Ala Ala 
 
11-20 GCT ATG AAG CGT CAC GGA CTT GAT AAC TAT 
 Ala Met Lys Arg His Gly Leu Asp Asn Tyr 
 
21-30 CGG GGA TAC AGC CTG GGA AAC TGG GTG TGT 
 Arg Gly Tyr Ser Leu Gly Asn Trp Val Cys 
 
31-40 GCT GCA AAA TTC GAG AGT AAC TTC AAC ACC 
 Ala Ala Lys Phe Glu Ser Asn Phe Asn Thr  
 
41-50 CAG GCT ACA AAC CGT AAC ACC GAT GGG AGT 
 Gln Ala Thr Asn Arg Asn Thr Asp Gly Ser 
 
51-60 ACC GAC TAC GGA ATC CTA CAG ATC AAC AGC 
 Thr Asp Tyr Gly Ile Leu Gln Ile Asn Ser 
 
61-70 CGC TGG TGG TGC AAC GAT GGC AGG ACT CCA 
 Arg Trp Trp Cys Asn Asp Gly Arg Thr Pro 
 
71-80 GGC TCC AGG AAC CTG TGC AAC ATC CCG TGC 
 Gly Ser Arg Asn Leu Cys Asn Ile Pro Cys 
 
81-90 TCA GCC CTG CTG AGC TCA GAC ATA ACA GCG 
 Ser Ala Leu Leu Ser Ser Asp Ile Thr Ala 
 
91-100 AGC GTG AAC TGT GCG AAG AAG ATC GTC AGC 
 Ser Val Asn Cys Ala Lys Lys Ile Val Ser 
 
101-110 GAT GGA AAC GGC ATG AAC GCG TGG GTC GCC 
 Asp Gly Asn Gly Met Asn Ala Trp Val Ala 
 
111-120 TGG CGC AAC CGC TGC AAG GGT ACC GAC GTC  
 Trp Arg Asn Arg Cys Lys Gly Thr Asp Val 
 
121-129  CAG GCG TGG ATC AGA GGC TGC CGG CTC TAG 
  Gln Ala Trp Ile Arg Gly Cys Arg  Leu Stop  
  
 
Note:  CTG  TAA  GTC   to CTC TAG  AGC 
     Leu     Stop      Val      Leu     Stop      Ser