Table Of ContentAPPLICATIONS
OF ENZYME
BIOTECHNOLOGY
INDUSTRY -UNIVERSITY COOPERATIVE CHEMISTRY
PROGRAM SYMPOSIA
Published by Texas A&M University Press
ORGANOMETALLIC COMPOUNDS
Edited by Bernard L. Shapiro
HETEROGENEOUS CAT AL YSIS
Edited by Bernard L. Shapiro
NEW DIRECTIONS IN CHEMICAL ANALYSIS
Edited by Bernard L. Shapiro
APPLICATIONS OF ENZYME BIOTECHNOLOGY
Edited by Jeffery W. Kelly and Thomas O. Baldwin
CHEMICAL ASPECTS OF ENZYME BIOTECHNOLOGY: Fundamentals
Edited by Thomas O. Baldwin, Frank M. Raushel, and A. Ian Scott
DESIGN OF NEW MATERIALS
Edited by D. L. Cocke and A. Clearfield
FUNCTIONAL POLYMERS
Edited by David E. Bergbreiter and Charles R. Martin
METAL-METAL BONDS AND CLUSTERS IN CHEMISTRY
AND CAT AL YSIS
Edited by John P. Fackler, Jr.
OXYGEN COMPLEXES AND OXYGEN ACTIVATION BY
TRANSITION METALS
Edited by Arthur E. Martell and Donald T. Sawyer
APPLICATIONS
OF ENZYME
BIOTECHNOLOGY
Edited by
Jeffery W. Kelly and Thomas O. Baldwin
Texas A&M University
College Station, Texas
Springer Science+Business Media, LLC
Library of Congress Catalog1ng-in-PublIcat1on Data
Texas A & M University, IUCCP Symposium on Applications of Enzyme
Biotechnology (9th : 199Ό
Applications of enzyme biotechnology / edited by Jeffery W. Kelly
and Thomas 0. Baldwin.
p. cm. — (Industry-university cooperative chemistry program
sympos i a )
"Proceedings of the Texas A & M University, IUCCP Ninth Annual
Symposium on Applications of Enzyme Biotechnology, held March 18-21,
1991, in College Station, Texas"—T.p. verso.
Includes bibliographical references and index.
1. Enzymes—Biotechnology—Congresses. I. Kelly, Jeffery W.
II. Baldwin, Thomas 0. III. Title. IV. Series.
TP248.65.E59T47 1991
660' .634—dc20 91-41625
. CIP
Proceedings of the Texas A&M University, IUCCP Ninth Annual Symposium
on Applications of Enzyme Biotechnology, held March 18-21, 1991, in College Station, Texas
ISBN 978-1-4757-9237-9 ISBN 978-1-4757-9235-5 (eBook)
DOI 10.1007/978-1-4757-9235-5
© 1991 Springer Science+Business Media New York
Originally published by Plenum Press, New York in 1991
Softcover reprint of the hardcover 1st edition 1991
All rights reserved
No part of this book may be reproduced, stored in a retrieval system, o r transmitted
in any form or by any means, electronic, mechanical, photocopying, microfilming,
recording, or otherwise, without written permission from the Publisher
FOREWORD
The Industry-University Cooperative Chemistry Program (IUCCP) has
sponsored eight previous international symposia covering a range of topics of interest to
industrial and academic chemists. The ninth IUCCP Symposium, held March 18-21,
1991 at Texas A&M University was the second in a two part series focusing on
Biotechnology. The title for this Symposium "Applications of Enzyme Biotechnology"
was by design a rather all encompassing title, similar in some respects to the discipline.
Biotechnology refers to the application of biochemistry for the development of a
commercial product. Persons employed in or interested in biotechnology may be
chemists, molecular biologists, biophysicists, or physicians. The breadth of biotech
research projects requires close collaboration between scientists of a variety of
backgrounds, prejudices, and interests.
Biotechnology is a comparatively new discipline closely tied to new
developments in the fields of chemistry, biochemistry, molecular biology and medicine.
The primary function of Texas A&M University is to educate students who will be
appropriately trained to carry out the mission of biotechnology. The IUCCP
Symposium serves as an important forum for fostering closer ties between academia
and industry and exchanging ideas so important to this evolving area.
The topics that were discussed during this conference, include the oxidation of
alkanes by enzymes, protein folding, waste remediation, protein purification techniques,
and protein expression systems. These titles represent a smorgasbord of topics of
importance to the biotechnology industry. The manuscripts submitted point out not
only the tremendous progress made in each one of those areas, but also discuss the
challenges still facing the industry as a whole. Many of the problems facing the biotech
companies are the same problems that academic biochemists and molecular biologists
face on a daily basis. It was clear to all participants that general solutions to thorny
problems such as protein expression, waste remediation, and refolding recombinant
proteins could form the basis for very successful companies. This pioneering and
entrepreneurial spirit is what makes biotechnology so exciting and what attracts some of
the brightest people to this area.
We are deeply indebted to the IUCCP sponsoring companies Abbott Labs,
Hoechst-Celanese, Monsanto Chemical Company, BF Goodrich, Dow Chemical
Company for providing the necessary resources to carry out this endeavor.
v
The co-chainnen of the conference were Professor Thomas O. Baldwin, and
Frank M. Raushel of the Texas A&M University Chemistry Department. The program
was developed by an academic steering committee consisting of the co-chainnen and
members appointed by the sponsoring chemical companies Dr. James Burrington, BP
America; Dr. Robert Durrwater, Hoechst-Celanese; Dr. Barry Haymore, Monsanto
Chemical Company; Dr. Mehmet Gencer, BF Goodrich; Dr. Paul Swanson, Dow
Chemical Company; and Professor Arthur Martell, Texas A&M IUCCP Coordinator.
In closing, the organizers of the Ninth IUCCP Symposium must recognize the
contributions that have been made to the symposium by Mrs. Mary Martell, who dealt
with the innumerable details necessary for a successful symposium. Her pleasant nature
and efficiency are appreciated. Finally, we wish to thank the Texas A&M graduate
students who donated their time to ensure smooth operations.
Thomas O. Baldwin
Jeffery W. Kelly
vi
CONTENTS
Minisymposium on Diagnostic Therapeutic Applications of Radiolabeled Antibodies
Radiolabeled Antibodies: Introduction and Metal Conjugation
Techniques .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 1
Sally W. Schwarz and Michael J. Welch
Methods for the Radiohalogenation of Antibodies ........................ 15
Michael R. Zalutsky, Pradeep K. Garg, Ganesan Vaidyanathan, and Sudha Garg
Diagnosis and Therapy of Brain Tumors Utilizing Radiolabeled Monoclonal
Antibodies ............................................. 29
Herbert E. Fuchs, Michael R. Zalutsky, Gary E. Archer, and Darell D. Bigner
Selective Functionalization of Alkanes by Enzymes and Their Models
Oxygenation by Methane Monooxygenase: Oxygen Activation and Component
Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 39
Wayne A. Froland, Kristoffer K. Andersson, Sang-Kyu Lee, Yi Liu,
and John D. Lipscomb
Structure and Mechanism of Action of the Enzyme(s) Involved in Methane
Oxidation ............................................... 55
Howard Dalton
Studies of Methane Monooxygenase and Alkane Oxidation Model Complexes 69
Amy C. Rosenzweig, Xudong Feng, and Stephen J. Lippard
Relevance of Gif Chemistry to Enzyme Mechanisms 87
Derek H.R. Barton and Dario Doller
Protein Folding and Refolding for Commercially Important Proteins
Transthyretin Acid Induced Denaturation Is Required for Amyloid Fibril
Formation in Vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 99
Wilfredo Colon and Jeffery W. Kelly
Isolation and Characterization of Natural and Recombinant Cyclophilins 109
T.F. Holzman, S.W. Fesik, C. Park, and J.L. Kofron
Mutations Affecting Protein Folding and Misfolding in Vivo . . . . . . . . . . . . . . .. 129
Anna Mitraki, Ben Fane, Cameron Haase-Pettingell, and Jonathan King
Protein Folding: Local Structures, Domains, and Assemblies . . . . . . . . . . . . . . .. 137
Rainer Jaenicke
VII
Environmental Biotechnology
Applications of Controlled Pore Inert Materials as Immobilizing Surfaces
for Microbial Consortia in Wastewater Treatment ................. 153
Ralph J. Portier
Organophosphorus Cholinesterase Inhibitors: Detoxification by Microbial
Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 165
Joseph J. DeFrank
Applications of Molecular Biology Techniques to the Remediation
of Hazardous Waste . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . " 181
Burt D. Ensley
Dehalogenation of Organohalide Pollutants by Bacterial Enzymes and Coenzymes 191
Lawrence P. Wackett
Protein Processing-New Techniques
Immobilized Artificial Membrane Chromatography: Surface Chemistry
and Applications ........................................ 201
Charles Pidgeon, Craig Marcus, and Francisco Alvarez
Perfusion Chromatography: Recent Developments and Applications . . . . . . . . . .. 221
Noubar B. Afeyan, Scott P. Fulton, and Fred E. Regnier
High Performance Capillary Electrophoresis of Proteins and Peptides:
A Minireview .......................................... 233
Robert S. Rush
Genetic Alterations Which Facilitate Protein Purification: Applications
in the Biopharmaceutical Industry ............................ 251
Helmut M. Sassenfeld, Michael Deeley, John Rubero,
Janet C. Shriner, and Hassan Madani
Expression Systems-Exogenous Proteins
Bacillus subtilis: A Model System for Heterologous Gene Expression 261
Roy H. Doi, Xiao-Song He, Paula McCready, and Nouna Bakheit
Aspergillus niger var. awamori as a Host for the Expression
of Heterologous Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 273
Randy M. Berka, Frank T. Bayliss, Peggy Bloebaum, Daniel Cullen,
Nigel S. Dunn-Coleman, Katherine H. Kodama, Kirk J. Hayenga,
Ronald A. Hitzeman, Michael H. Lamsa, Melinda M. Przetak,
Michael W. Rey, Lori J. Wilson, and Michael Ward
Poxvirus Vectors: Mammalian Cytoplasmic-Based Expression Systems 293
Bernard Moss
Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 301
Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 303
viii
RADIOLABELED ANTIBODIES: INTRODUCTION AND METAL
CONJUGATION TECHNIQUES
Sally W. Schwarz and Michael J. Welch
Mallinckrodt Institute of Radiology
Washington University, 510 S. Kingshighway
St. Louis, MO, U.S.A.
INTRODUCTION
The use of radiolabeled antibodies in the detection and treatment of cancer has been in
practice since the early 1980's. Radioimmunoimaging is an in vivo diagnostic technique
where a radiolabeled antibody is taken up or bound to an antigen in a target tissue. This
allows for non-invasive imaging of the antigen containing tissue, using a gamma camera
or a positron emission tomograph (PET) scanner, for subsequent therapy or resection of
the tissue if necessary. Radioimmunotherapy is the delivery of a therapeutic quantity of
a radioisotope to the same antigen containing tissue to ablate or reduce a primary or
metastatic carcinoma. This chapter will cover the basic principles of antibodies,
subsequent conjugation with bifunctional chelates and radiolabeling for the purpose of
radioimmunoimaging or radioimmunotherapy.
Antibodies (Ab) are immunoglobulins produced as a result of the body's immune
response. This reaction is triggered when the body is faced with foreign matter.
"Immunogens" or antigens can be bacteria, viruses, fungi, or any foreign proteins.
Each antigen (Ag) has more than one epitope or antigenic determinant (figure 1). These
epitopes represent only a small fraction of the Ag molecule.
Once a single Ag is injected into the bloodstream it interacts either by direct associaton
with a B-Iymphocyte or the Ag is "processed" by macrophages and "presented" to the B
lymphocyte by a T-helper cell. After interaction with the B-Iymphocyte the B-cell is
activated, and proliferates. It then differentiates into a plasma cell which secretes the Ah
specific for the Ag (figure 2). After Ag injection there is a lag time of approximately one
week before production of these Ag specific Ab.
Antibodies are glycoproteins consisting of five different classes: IgG, IgM, IgA, IgE and
IgD (figure 3). Each group contains one or more subunits of a Y shape. Each Y unit
contains 4 polypeptides, two identical segments called heavy chains and 2 segments called
light chains (figure 4). Each class of Ab has a specific type of heavy chain, but there are
only 2 types of light chain polypeptides known as kappa (lC) and lambda ().). These chains
are held together with multiple inter and intra disulfide bonds and non-covalent bonds.
When the body is faced with an unknown Ag, the IgM class of immunoglobulins is
