Table Of ContentINVESTIGATION OF RECEPTOR BINDING SITES IN HUMAN TRANSFORMING
GROWTH FACTOR ALPHA
By
SUSAN BRITA RASMUSSEN
r
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1997
.
ACKNOWLEDGMENTS
I would like to thank everyone whose help and friendship
made graduate school a joy. In addition, I would like to thank
my mother, who showed me that I could choose any career and
succeed, my dad, who showed me how to achieve my goals, my
brother, who taught me that anything is possible, and my fiance,
who believed in my ability and kept me sane and happy during
graduate school.
Next, I would like to thank my research professor, Dr. Nigel
Richards, for allowing me to work and learn from him. I thank my
other family, the Richards research group. Working with them was
a pleasure and their friendship is immeasurable. I thank my
friend and teacher Regina Shaw who taught me all I know about the
world of molecular biology with wit and wisdom. I also give my
heartfelt gratitude to my good friend and mentor Dr. William
Farmerie who taught me how to do research and notice the little
details. I would also like to thank Dr. Gregory Schultz for his
helpful suggestions and comments.
Last but not least, I thank my best friends, Karen Frank,
Larry Land and Lisa Spurlock. It was a joy finding them during
graduate school; their friendship and love made graduate school
enjoyable
TABLE OF CONTENTS
page
ACKNOWLEDGMENTS ii
LIST OF TABLES vi
LIST OF FIGURES vii
ABBREVIATIONS ix
ABSTRACT X
CHAPTERS
1 TRANSFORMING GROWTH FACTOR ALPHA AND ITS
INTERACTION WITH THE EPIDERMAL GROWTH FACTOR
RECEPTOR 1
Introduction 1
Transforming Growth Factor Alpha 3
Epidermal Growth Factor Receptor 5
EGF-R Tyrosine Kinase Activity 6
EGF Receptor Signaling and Dimerization 7
Human Transforming Growth Factor Alpha Structure ... 8
X-ray Crystallography of hTGFa 11
Nuclear Magnetic Resonance Structures of hTGFa . 11
Peptide Mapping and Site Directed Mutagenesis
of TGFa 12
Specific Aims 15
2 EXPERIMENTAL METHODS AND MATERIALS 20
Materials 20
Enzymes 20
Oligonucleotides 20
Methods 20
Sodium Dodecyl Sulfate Polyacrylamide Gels
(SDS-PAGE) 20
DNA Preparation 21
Ligation and Transformation Reactions 21
Bacterial Strains and Vectors 21
pHisrrxApET22b(+) [pHisTrx] Vector Assembly .... 22
DNA Sequencing 22
Recombinant TGFa Gene Construction 22
page
CHAPTERS
Expression of Recombinant hTGFa Protein in
ExpprEeTs-s2i2obn(+o)f Recombinant HisTrxA-hTGFa 26
Protein 27
HisTrxA Fusion Protein Purification 27
Enterokinase Cleavage of Fusion Protein 28
Purification of hTGFa 28
hTGFa Antiserum Production 29
Enzyme Linked Immunosorbant Assay of hTGFa
Polyclonal Antibody 30
Western Immunoblot Analysis of hTGFa Protein ... 31
hTGFa lodination Protocol 31
Human Placenta Homogenization 32
Radiolmmuno- and RadioReceptor- Assays of
hTGFa Protein 33
Cell Culture 34
Methyl'^H-Thymidine Incorporation Assay 35
Protein Concentration Determination 36
3 PROTEIN EXPRESSION SYSTEMS 37
Introduction 37
Eukaryotic Expression Systems 37
Prokaryotic Expression Systems 40
Chemical Synthesis of hTGFa 41
Preliminary hTGFa Protein Expression Efforts 42
Expression Vector Selection 42
Native hTGFa and Mutant hTGFa Gene Assembly .... 45
Method Development for hTGFa Protein
Expression 45
Purification of Expressed pelB-hTGFa Protein ... 47
Enterokinase Cleavage of pelB-hTGFa Protein .... 49
RIA/RRA Analysis of Expressed Proteins 50
4 FINAL EXPRESSION SYSTEM AND PURIFICATION 53
Introduction 53
Histidine Tag 54
Thioredoxin 55
HisTrxA-hTGFa Fusion Protein Expression 56
HisTrxA-hTGFa Fusion Protein Purification 56
5 VALINE 33 HINGE MUTANTS 67
The Hinge Region of hTGFa and Its Relevance 67
Receptor Binding and Activation of the Val33
Mutants 75
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page
CHAPTERS i
6 PHENYLALANINE 15 MUTATIONS 80
Biological Significance of Phel5 in hTGFa 80
Receptor Binding Affinity of Phel5 Mutants 86
7 CONCLUSIONS AND FUTURE WORK 89
Conclusions 89
Future Work 91
Enterokinase Cleavage Reaction 91
NMR Structural Analysis of hTGFa Mutants 92
Dimerization Event Analysis 94
Future hTGFa Mutants 97
Use of Different Receptor Sources 99
BIBLIOGRAPHY 103
BIOGRAPHICAL SKETCH Ill
V
LIST OF TABLES
Table page
1-1 Sequence Alignment of Epidermal Growth Factor Family
Members 2
2-1 hTGFa DNA Oligonucleotide Primers 24
3-1 pelB-hTGFa Crude Expression Yield 51
4-1 HisTrxA-hTGFa Crude Expression Yield 58
4-2 Purified Wildtype Recombinant HisTrxA-hTGFa
Activity 61
4-3 Contribution to Binding Affinities from HisTrxA
Fusion Protein 66
5-1 Alignment in Valine 33 Region of EGF Family Members.. 68
5-2 Mutations Done in the Hinge Region of hEGF and
hTGFa 69
5-3 Comparison of Calculated Concentrations from
OD280Values and RIA Values 75
5-4 Relative Binding Affinities of Val33 Mutants 76
5-5 Mitogenic Stimulation Capability of Valine 33
Mutants 77
6-1 Alignment in Phenylalanine 15 Region of EGF Family
Members 82
6-2 Previous Mutations at Position 13 of hEGF and
Position 15 of hTGFa 82
6-3 Comparison of Calculated Concentrations from
OD280 Values and RIA Values 83
6-4 Relative Binding Affinities of Phel5 Mutants 87
6-5 Mitogenic Stimulation Capabilities of Phel5
Mutants 88
vi
LIST OF FIGURES
Figure page
1-1 EGF Receptor Domains 6
1-2 EGF Receptor Dimerization Model 9
1-3 Structure of Human TGFa 10
1-4 Side View of hTGFa 17
1-5 Front View of hTGFa 18
1-6 Rear View of hTGFa 19
2-1 Overlap PGR Mutagenesis 25
3-1 Structure of Human Transforming Growth Factor a 38
3-2 pET-22b(+) Expression Vector 43
3-3 Overlap PGR Mutagenesis 46
3-4 SDS-Page Gel of pelB-hTGFa 47
3-5 Western Immunoblot of peiB-hTGFa 48
3-5 Western Immunoblot of DEAE Column Fractions
Containing pelB-hTGFa 49
3-7 DEAE Column Trace of pelB-hTGFa and hTGFa 52
3-8 DEAE Column Trace of pelB-hTGFa and Periplasm 52
4-1 Coordination of Nickel with a Histidine Containing
Protein 55
4-2 pHisTrxA Expression Vector 59
4-3 SDS-Page Gel Analysis of HisTrxA-hTGFa Expression.... 60
4-4 Western Immunoblot of HisTrxA-hTGFa Expression 60
4-5 SDS-PAGE Gel of Metal Chelate Affinity Column
Fractions Containing HisTrxA-hTGFa 61
vii
Figure page
4-6 HPLC Trace of Recombinant hTGFa 62
4-7 HPLC Coinjaction of Recombinant hTGFa and Commercial
hTGFa 63
4-8 Western Immunoblot of Enterokinase Nonspecific
Degradation 64
4-9 SDS PAGE Gel of Enterokinase Cleavage Reactions of
HisrrxA-hTGFa Protein at pH7 and pH8 64
4-10 SDS-Page Gel of the Stability of HisTrxA-hTGFa
at 37°C 65
4-11 Scatchard Plot of HisTrxA+TGFa and TGFa Compared
with the TGFa Standard 66
5-1 Chemical Structures of Valine 33 Substitutions 70
5-2 Standard Curve of DNA Stimulation by hTGFa 74
5-3 Relative Binding Affinity Graph of Val33 Mutants 76
5-4 Mitogenic Stimulation of Val33 Mutants (Trial 1) 77
5-5 Mitogenic Stimulation of Val33 Mutants (Trial 2) 78
6-1 Chemical Structures of Phel5 Substitutions 84
6-2 Relative Binding Affinities of Phel5 Mutants 87
6-3 Mitogenic Stimulation of PhelS Mutants 88
viii
)
ABBREVIATIONS
amp ampicillin
AU absorption units
C carboxyl
Ca alpha carbon
d deoxy
E. coli Escherichia coli
EDTA ethylenediaminetetraacetate
EGF Epidermal Growth Factor
HCl hydrochloric acid
HPLC high performance liquid chromatography
hTGFa Human Transforming Growth Factor Alpha
IPTG isopropylthio-p-D-galactopyranoside
kDa kilodalton
LB Luria Bertani Media
mg milligram
mmLM(s) mmiilllliilmiotlearr(s
MWCO molecular weight cut off
N amino
NaCl sodium chloride
ng nanogram
nM nanomolar
nm nanometer
OD optical density
PAGE polyacrylamide gel electrophoresis
SDS sodium dodecyl sulfate
TGFa Transforming Growth Factor Alpha
TFA trifluoroacetic acid
Tricine N-[Tris-hydroxymethyl Methyl] glycine
Tris tris (hydroxymethyl) a)minomethane
^g microgram
f^M micromolar
ix
i
Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
INVESTIGATION OF RECEPTOR BINDING SITES IN HUMAN TRANSFORMING
GROWTH FACTOR ALPHA
By
Susan Brita Rasmussen
May, 1997
Chairman: Dr. Nigel G.J. Richards
Major Department: Chemistry
Human transforming growth factor alpha (hTGFa) is
implicated in the progression of tumor cell vascularization,
ultimately leading to metastasis. hTGFa is a member of the
epidermal growth factor family, and binds to the EGF-receptor
(EGF-R). This research has focused on testing several hypotheses
concerning the roles of specific residues and regions of the
protein in EGF-R binding and stimulation. Previous work had
shown that bacterial expression of hTGFa resulted in the
formation of insoluble inclusion bodies, limiting the ability of
such systems to yield large amounts of correctly folded hTGFa.
Given the technical simplicity of generating recombinant proteins
in bacteria, the development of a new expression system for hTGFa
in Escherichia coli was investigated. The first construct
employed the commercial vector, pET22b(+) (Novagen, Madison, WI),
in which hTGFa expression is regulated under the tightly
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