Preface such as Escherichia coli for the production of recombinant proteins has many limitations. One set of problems relates to the intracellular accumulation of the product: The total achievable product titer is limited by the achievable cell density; the protein is frequently produced in a denatured state and therefore requires subsequent costly and inefficient renaturation; and the bacterial membrane has to be ruptured (or the whole cell has to be broken) to release the protein, complicating downstream purification procedures. An additional problem associated with intracellular accumulation of proteins is the requirement that the protein be compatible with the host cell. This requirement further restricts the range of proteins that can be produced by a given bacterial host. A problem associated with the use of procaryotic bacterial hosts, such as E. coli, is the inability to glycosylate proteins. Although many proteins do not require glycosylation for activity, proteins originating in higher life forms (such as animal, insect, and plant tissues) often require glycosylation. Finally, for the product to be acceptable for use in foods and beverages, the host bacteria should be generally recognized as safe (GRAS), which is not the case for E. coli, to ease regulatory approval of the production process. For many reasons, including those just given, it has become important to expand the range of hosts available for production of products. The rapid development of the tools of genetic engineering for the manipulation of cellular DNA has provided the necessary capability to develop a number of successful applications in an expanding range of expression systems. Expression systems, which originated in procaryotic bacteria with internally accumulated proteins encoded by bacterial DNA, have evolved to increasingly complex, higher life forms. It is now possible to produce proteins encoded by the DNA of foreign organisms in a wide variety of procaryotic and eucaryotic cells (including yeast, insect, plant, and animal cells). The technology of genetic engineering has also led to routine use of new expression systems for the synthesis and secretion of proteins in prokaryotic microorganisms with DNA originating from eukaryotic cells. This technology includes production systems capable of yielding high concentrations of secreted proteins ( 1 - 2 g/L) in GRAS hosts. It has also led to a number of production systems capable of glycosylating proteins.
Expression Systems and Processes for rDNA Products Downloaded from pubs.acs.org by YORK UNIV on 12/11/18. For personal use only.
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Since in vitro manipulation of DNA through the use of polymerase chain reaction (PCR) was developed by Cetus, the utility of recombinant expression systems for the production of useful proteins has been signifi cantly expanded. With PCR, it is now possible to modify DNA rapidly to alter the structure and properties of proteins to suit specific applications. The ease of production of test quantities of the new proteins then allows the determination of the structure-function relationship in the environ ment of the application and the subsequent development of proteins with properties optimally suited to the end use. This capability, coupled with the availability of a wide range of expression systems, is leading to an ever-increasing range of applications, including those in the pharmaceuti cal, food, and chemical industries. To improve future production systems further, it will be necessary to determine the dynamics of the intracellular processes. The rate-limiting steps are still inadequately characterized to permit full optimization of the production and secretion of proteins. It is also necessary to understand the energetics of intracellular processes further to allow optimization of small molecule production. The purpose of this volume is to report on recent developments in new expression system technologies as well as relevant process technol ogy. The chapters discuss bacterial hosts (E. coli), yeast (Saccharomyces cerevisiae), and insect cells. The process technologies included are highcell-density bacterial fermentations, biocatalysis, and process issues with recombinant microorganisms. Overall, the book represents the range of activities under way in the development and use of recombinant microor ganisms and tissues for advanced processes and products. Acknowledgments We express our appreciation to the authors for their contributions to this book. We also thank the Division of Biochemical Technology for spon soring the symposia represented by these papers. Finally, we are espe cially appreciative to the editorial staff of the ACS Books Department and to Robin Giroux. RANDOLPH T. HATCH
ANTONIO MOREIRA
Aaston, Inc. 12 Falmouth Road Wellesley, MA 02181
University of Maryland TRC, Room 250 Baltimore, MD 21228
CHARLES G O O C H E E
YAIR
Department of Chemical Engineering Stanford University Stanford, CA 94305-5025
Schering-Plough Corporation 1011 Morris Avenue
September 2, 1991
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ALROY
Union, NJ 07083
