Review
Recombinant protein expression for therapeutic applications

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Abstract

In recent years, the number of recombinant proteins used for therapeutic applications has increased dramatically. Many of these applications involve complex glycoproteins and antibodies with relatively high production needs. These demands have driven the development of a variety of improvements in protein expression technology, particularly involving mammalian and microbial culture systems.

Introduction

As the biotechnology industry has rapidly expanded in recent years, the expression of a spectrum of recombinant proteins in different systems for a wide variety of purposes has been a major feature and challenge. In some applications, a large array of proteins are needed in relatively small quantities for screening applications, whereas in other cases, quantities approaching the metric ton scale are needed for specific therapeutic applications. The majority of therapeutic proteins have been produced in either mammalian cell-culture systems, with Chinese hamster ovary (CHO) cells representing the most common system, or in Escherichia coli 1., 2•.. A variety of alternative expression systems are also being developed and evaluated. It is not at all clear which of these systems will ultimately be the most useful for the variety of niches and applications of therapeutic protein production.

Several excellent reviews have provided comprehensive coverage of various aspects of therapeutic protein production, including specific issues related to large-scale cell culture production [1] and considerations for the production of specific classes of molecules such as recombinant antibody-related products [3]. This review will focus on selected results across a range of expression systems (although focusing primarily on mammalian and E. coli cell-culture systems) that illustrate important ways in which expression technology is evolving to meet the spectrum of research, development and commercial needs. Rather than provide a comprehensive review of the subject, this review will highlight several specific, recent results across the field.

Section snippets

Mammalian expression systems

Over the past seven to eight years there has been considerable progress in fine-tuning mammalian expression systems for high-level recombinant gene expression. CHO and NS0 murine myeloma cell expression systems have now established themselves as the predominant systems of choice for mammalian expression. Refinements of vector construction, choice of selectable markers and advances in gene-targeting and high-throughput screening strategies have made the establishment of recombinant cell lines

Microbial expression systems

The primary microbial host for producing recombinant therapeutic proteins has been E. coli, and recent reviews have provided excellent summaries of key elements of this topic 2•., 43..

In many cases, the production of soluble, active protein is desired (as opposed to inclusion-body formation), and a variety of cases where proteins with chaperone-like activity have enhanced folding have been recently reported. DegP represents one interesting case as it has been demonstrated to have chaperone-like

Other expression systems

Both transgenic plants and animals, as well as plant tissue cultures, have been used to produce a variety of different recombinant proteins (reviewed in 73., 74., 75.). Whereas limits in glycosylation have been cited as one challenge for recombinant protein production in plants, the problem of proteolysis was also evaluated in a recent report on the potentially important application of antibody production in plant culture [76••].

Insect cells have been used in a variety of protein expression

Linking expression and purification

Increasing attention has been paid to optimizing expression systems in the context of the production process to aid in protein recovery and to improve overall yields and efficiencies. For example, the complementary expression of holin and lysozyme enzymes in E. coli to facilitate the release of products has been demonstrated for two different cases 91., 92., 93. The co-expression of an endonuclease was also used to improve the properties of the lyzed fermentation broth for subsequent processing

Conclusions

In recent years, a variety of different platforms have been investigated for the production of recombinant therapeutic proteins. CHO and NS0 murine myeloma cells are the predominant mammalian cell lines used, and recent progress has been made in promoter systems and in the manipulation of these cell lines to improve glycoprotein production in fermentors. Rational engineering of E. coli has also continued, with notable advancements in improving the cellular environment for protein folding.

Acknowledgements

The authors would like to thank John Joly, Brad Snedecor and Gian Polastri for discussions and critical review of the manuscript.

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

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