PROTEIN CO-CRYSTALLIZATION RECOMBINANT ANTIBODY FRAGMENT LIBRARY ...

174 Pages·2003·1.69 MB·English

PROTEIN CO-CRYSTALLIZATION RECOMBINANT ANTIBODY FRAGMENT
LIBRARY BIOTECHNOLOGY
by
LAURA-LEE CLANCY KELLEY
(Under the Direction of Professor Cory Momany)
ABSTRACT
Co-crystallization recombinant antibody fragments (crFabs) are antibody
fragments derived from a parent antibody fragment that can be used to assemble a 3-
dimensional matrix (crystal). Our hypotheses were the following:
1. A molecule of known structure can be modified by genetic recombination to draw
another macromolecule along with it into a crystal matrix.
2. The unknown structure can be determined with the aid of the known component.
We have the ability to determine macromolecular structures rapidly. A rate-
limiting step is frequently at the crystal formation stage. Typically, only about 20% of
macromolecules screened with a large variety of reagents by any available crystallization
strategy produce usable crystals. With the development of crFab-based crystallization,
those odds may be improved considerably.
Four Fab mutants of a mouse recombinant antibody fragment were tested for their
suitability as co-crystallization reagents. The first mutant had a 6-His purification tag on
the C-terminus of the heavy chain of the Fab (Fab2). Since Fab2 did not crystallize, the
second mutant was modified to have four vicinal histidines, two on each chain. It was
discovered that this mutant had a leucine residue inserted into a beta-pleated sheet when
compared with mFab25.3. Fab4 was then constructed by a deletion mutation of that
residue. Finally a heavy chain (N57→K) mutation was created to engineer a stronger salt
bridge between the Fab molecules as they packed into the crystal structure (Fab5).
Mutant rFab4 proved to be the best mutant for co-crystallization studies based on its yield
and crystallization properties. By multi-site directed mutagenesis of the third
hypervariable regions of the light and heavy chains, a recombinant Fab antibody
fragment library was built with a nominal diversity of 1.16 x 107 colony forming units.
This library was shown to produce rFabs that could recognize seven out of seven proteins
tested and has the potential for being used for the co-crystallization of proteins.

INDEX WORDS: Antibody fragment library, Multi-site directed mutagenesis,
Biotechnology, Crystallization
CO-CRYSTALLIZATION RECOMBINANT MOUSE ANTIBODY FRAGMENT
LIBRARY BIOTECHNOLOGY

by

LAURA-LEE CLANCY KELLEY

B.S., Michigan State University, 1972
A. A. S. Nursing, Community College of Allegheny County, 1982
M.S., University of Pittsburgh, 1986

A Dissertation Submitted to the Graduate Faculty of The University of Georgia in Partial
Fulfillment of the Requirements for the Degree

DOCTOR OF PHILOSOPHY

ATHENS, GEORGIA
2003
© 2003
Laura-Lee Clancy Kelley
All Rights Reserved
CO-CRYSTALLIZATION RECOMBINANT MOUSE ANTIBODY FRAGMENT
LIBRARY BIOTECHNOLOGY

by

LAURA-LEE CLANCY KELLEY

Major Professor: Cory Momany

Committee: Bi Cheng Wang
E. W. Taylor
N. G. Newton
A. Capomacchia
Mark Eiteman

Electronic Version Approved:

Maureen Grasso
Dean of the Graduate School
The University of Georgia
May 2003
DEDICATION
I would like to dedicate this dissertation to my husband, David D. Kelley, for his
constant encouragement, supportiveness and faithful devotion. Also, it is dedicated to the
memory of my father who was proud to have me for his daughter and encouraged all of
his children to follow after whatever God put in their hearts to do.

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ACKNOWLEDGEMENTS
I would like to acknowledge those who have helped me with my academic career.
My high school physics teacher, Mr. Ralph Facziska, made physics understandable,
interesting and practical, by showing me a side of science that was the exact opposite of
the attitudes expressed by most of my peers. Also, I will be forever grateful to the Dean
of the Faculty of Arts and Sciences at the University of Pittsburgh, Jerald Rosenberg, for
his encouragement while I was an undergraduate student and his friendship over the years
and to Professor John Rosenberg for introducing me to the science of crystallography.
Professor Bi-Cheng Wang, affectionately known as “B.C.” to all his students and
colleagues, has been a source of inspiration by promoting my career as a student here at
the University of Georgia as well as having been the chairman of my master’s committee
at the University of Pittsburgh. He is an excellent teacher of protein crystallography. His
inclusion of us at his parties has been helpful in many ways.
Professor Cory Momany has been a wonderful source of advice and an excellent
director of the progress I have made in my research on the topic discussed in this
dissertation and for his instruction in the use and maintenance of laboratory equipment
that was new to me. He has willingly been “on call” for late evening questions regarding
instrument problems every time they arose. I have enjoyed working with him for the past
four and a half years.
Professor Anthony Capomacchia has truly been a student advocate for me as well
as others.
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Professor E. William Taylor has graciously allowed me to use his PCR equipment
and has also spoken encouragingly to me at times as well as contributing to committee
meetings in such a way that made me feel he was on my team.
Professor Emeritus Gary Newton has also provided insightful comments. I have
appreciated having him on my committee. Every member of the committee has been
wonderful to me and a great group to know.
Professor Mark Eiteman in the Department of Biological & Agricultural
Engineering provided very useful conversations on bioreactor processes and graciously
allowed me to use his facilities. I appreciate his willingness to join the committee at this
late hour. Geoffrey Smith and Sarah Lee were also helpful in Dr. Eiteman’s lab for
assisting to set up the bioreactor experiments. Atin Tomar was particularly helpful when
he took the late night samples on the second bioreactor process.
I would also like to express my appreciation of the people I have worked along
side of in the Momany lab in the last four years: the Russian post-doctoral fellows Elena
Blagova and Vladimir Levdikov, especially for helping me with the HPLC instrument
and their rallying support when I needed help to move from one apartment to another,
and fellow graduate students, Nandita Bose, Ph. D., Alex Vandell, Pharm. D., and Jay
Houston, for their encouragement and help both professionally and personally. Various
undergraduate students have contributed to the diversity of the lab, but Betty Ngo was an
outstanding technician and Avis Scott shows great potential as a lab worker.
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TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS..............................................................................................v
LIST OF TABLES.............................................................................................................x
LIST OF FIGURES.........................................................................................................xi
CHAPTER
1 BACKGROUND AND SIGNIFICANCE......................................................1
1.1 Overview.................................................................................................1
1.2 Crystallization of Proteins....................................................................2
1.3 Antibodies, Types of Antibody Fragments and their Structure.......6
1.4 A Brief History of Biotechnology and Antibodies ............................10
1.5 Phage Display Mechanism in Escherichia coli..................................12
1.6 Significance..........................................................................................15
2 THE CURRENT STATE OF THE ART OF RECOMBINANT
ANTIBODY FRAGMENT BIOTECHNOLOGY..................................17
2.1 Overview...............................................................................................17
2.2 Problems Associated with Recombinant Antibody Fragment
Biotechnology.......................................................................................17
2.3 Applications of Antibody Fragment Biotechnology.........................23
2.4 Future Applications and Developments in Biotechnology...............33
2.5 Summary..............................................................................................37
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3 OVEREXPRESSION, PURIFICATION AND CHARACTERIZATION
OF MOUSE RECOMBINANT ANTIBODY FRAGMENTS IN
ESCHERICHIA COLI...............................................................................38
3.1 Overview...............................................................................................38
3.2 Rationale...............................................................................................39
3.3 Materials and Methods.......................................................................40
3.4 Results...................................................................................................51
3.5 Discussion.............................................................................................74
4 MOUSE RECOMBINANT ANTIBODY FRAGMENT PRODUCTION
IN A BIOREACTOR................................................................................76
4.1 Overview...............................................................................................76
4.2 Rationale...............................................................................................76
4.3 Materials and Methods.......................................................................77
4.4 Results...................................................................................................81
4.5 Discussion.............................................................................................87
5 GENERATION OF A PHAGEMID MOUSE RECOMBINANT
ANTIBODY FRAGMENT LIBRARY BY MULTI-SITE DIRECTED
MUTAGENESIS ......................................................................................89
5.1 Overview...............................................................................................89
5.2 Rationale...............................................................................................90
5.3 Materials and Methods.......................................................................91
5.4 Results...................................................................................................99
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