Protein Expression Technologies: Current Status and Future Trends Chapter Abstracts
Chapter 1
Expression of Extremophilic Proteins
Amy M. Grunden, Donald A. Comfort, Erica L. Malotky, and Robert M. Kelly
Proteins from extremophilic microorganisms, those inhabiting niches which are characterized by biologically-extreme temperatures, ionic strengths, pressures, and/or pHs, have been examined for their scientific merit and biotechnological potential. Difficulties encountered in obtaining the native forms of these proteins have fostered efforts to express recombinant versions in foreign hosts and, in some cases, in the extremophile itself. While proteins from a variety of extremophiles have been successfully produced in recombinant forms, there are significant problems that can be encountered that go beyond those normally found with recombinant protein production. Overcoming these problems is key to future scientific efforts as well as to use of extremophilic proteins in technological applications.
Chapter 2
Expression, Folding and Degradation in Escherichia coli
Mirna Mujacic and François Baneyx
The Gram-negative bacterium Escherichia coli has been and remains extensively used for the production of heterologous proteins at both laboratory and industrial scales. Over the past decade, sophisticated genetic tools and a growing understanding of the function of the E. coli proteome have been exploited to improve the synthesis of complex proteins of therapeutic or commercial interest in a soluble and biologically active form. This chapter reviews the chief aspects of heterologous protein production in E. coli with a focus on the mechanisms of folding, degradation and export, and the practical implications of recent discoveries in these areas.
Chapter 3
Tools for Metabolic Engineering in Escherichia coli
Christina D. Smolke, Vincent J.J. Martin, and Jay D. Keasling
Metabolic engineering is the redirection of cellular metabolism for the production of valuable or novel chemicals or to remediate toxic chemicals in the environment. To genetically modify cellular metabolism, particular gene expression tools are required for the precise and balanced expression of multiple genes, so that flux through a heterologous pathway is not limited by any one enzyme and cellular resources are not wasted. These tools target many aspects of gene expression control and host development including stable maintenance of heterologous DNA in a host cell (chromosome engineering, low-copy number plasmids); stringent, homogeneous, and timed control over gene expression (promoter design); coordinated and differential production of multiple enzymes in a pathway (directed RNA processing and decay, translational control); techniques to generate genetic diversity (directed evolution strategies); and tools that enable the quantification of metabolism and biochemical pathways (genome-wide analytical tools, metabolic modeling). This chapter reviews the current tools available for metabolic engineering applications in E. coli and points to potential areas where further developments are needed.
Chapter 4
Expression Systems in Bacillus
Rob Meima, Jan Maarten van Dijl, Siger Holsappel, and Sierd Bron
Bacillus subtilis is the best-studied Gram-positive bacterium known today. Over the past five decades this species has become the paradigm of Gram-positive genetics and physiology. In addition, B. subtilis and several other bacilli have a long history of safe use in traditional food production and industrial scale applications, making them of considerable commercial importance. In this chapter, we describe some salient features of gene expression in this intriguing family of microbes. A general overview of the genus, some of its members and their characteristics is presented first. The next section outlines general characteristics of gene expression, and highlights a number of examples of the expression and regulation repertoire available to the Bacillus cell. A section on protein secretion describes the different, well-characterized protein secretion pathways of bacilli, which are capable of secreting a large number of different proteins, often at considerable levels. Next, an overview of commonly used vectors and (inducible) promoters is given, followed by a section devoted to Bacillusgenomics as an important source of new insights and tools. The chapter concludes with a summary of current topics in the field of Bacillusresearch and perspectives for future developments.
Chapter 5
Saccharomyces cerevisiae Protein Expression: from Protein Production to Protein Engineering
Ronald T. Niebauer and Anne Skaja Robinson
The yeast Saccharomyces cerevisiae has long been recognized and used as a host for protein expression since it can offer the processing steps of mammalian systems along with the ease of use of microbial systems. Here, some of the factors enabling effective protein expression are highlighted including vector design, expression strategies, potential problems encountered and methods to deal with these problems. Current and future challenges of the Saccharomyces cerevisiae system are also addressed. Emerging technologies based on functional studies, which can be applied to applications such as drug discovery, protein engineering for enhanced properties, and functional genomics are addressed. The versatility associated with using Saccharomyces cerevisiae as a tool to address these current and future challenges should allow a continued role in protein production, and in understanding and engineering proteins as well.
Chapter 6
Production of Recombinant Proteins Using the Methylotrophic Yeasts Pichia pastoris and Pichia methanolica
Bruce L. Zamost, Gary J. Lesnicki and Sanjay Jain
Pichia expression systems have been used to produce a wide variety of mammalian, microbial, and plant proteins. The methylotrophic yeasts Pichia pastoris and Pichia methanolica are capable of high level expression when heterologous genes are placed under transcriptional control of the methanol-inducible alcohol oxidase (AOX1 or AUG1) promoters. Additional inducible and constitutive promoters have also been used for recombinant protein synthesis. Pichia systems have a number of desirable features: (1) the yeast host strains are readily amenable to genetic manipulations; (2) genetic stability can be ensured by integrating expression cassette(s) into the host chromosome; (3) cultures readily adapt to high-biomass fermentations in low-cost, defined medium; (4) high level expression with disulfide bond formation is possible; and (5) Pichia can perform specific post-translational modifications.
Chapter 7
Advances in Protein Expression in Filamentous Fungi
Hendrik J. Meerman, Aaron S. Kelley, and Michael Ward
Filamentous fungi have a long history of safe industrial cultivation for the efficient production of a diverse set of compounds, including proteins, organic acids and secondary metabolites such as antibiotics, carotenoids, and vitamins. In particular, both endogenous and exogenous enzymes are currently manufactured commercially using fungal host systems. The advantages of recombinant protein expression in filamentous fungi have been detailed extensively over the years, such as in several recent excellent reviews (Punt et al., 2002; Bergquist et al., 2002; Archer, 2000). In this chapter we will recapitulate some of the highlights featured in those reviews and emphasize some recent advances that have significantly improved the understanding and utilization of fungal hosts. Specifically, we will give examples of the different strategies used to produce various proteins in fungi at Genencor, and a perspective on improvements that are in progress in other laboratories. We will also address the regulation of expression and secretion of foreign proteins, and discuss the effect of fermentation technology on protein production in fungi.
Chapter 8
Insect Cell Expression Systems
Karthik Viswanathan, Yu-chan Chao, John T. A. Hsu, and Michael J. Betenbaugh
Insect cells in combination with infectious baculoviruses have been used for the production of numerous heterologous proteins. This expression system relies on the generation of recombinant baculoviruses in which viral genes active during the late phases of infection, but not essential for viral replication in cell culture, are replaced by DNA sequences of interest. Viral deletions and specific molecular markers ensure efficient production and selection of recombinant baculoviruses. The high strength of the polyhedrin and p10 promoters enables high level expression of target proteins starting approximately 20 hours post-infection and lasting until cell lysis, three to five days thereafter. Direct incorporation of foreign genes into the host chromosome and the development of non-lethal viruses can eliminate cell death that accompanies traditional baculovirus vector systems. Insect cell lines are being isolated and engineered to include complex post-translational processing events similar to those of mammalian cells in order to produce heterologous glycoproteins that are virtually identical to those generated by mammalian hosts. Insect larvae are also being utilized as a more economical method for production of recombinant proteins of interest. The convenience and flexibility of the baculovirus system has led to its expanding use in other areas. Display on the baculovirus surface is being used to evolve proteins with altered properties and as a tool to present antigens. Baculovirus vectors are also being evaluated as gene delivery vehicles to express foreign genes in mammalian cells and other non-permissive hosts. These developments will ensure that insect cells continue to be a versatile and widely used expression system for the foreseeable future.
Chapter 9
Heterologous Expression of Genes in Chinese Hamster Ovary Cells
Gyun Min Lee and Sun Ok Hwang
For the production of therapeutic proteins, recombinant Chinese hamster ovary (rCHO) cells with dihydrofolate-reductase (DHFR)-mediated gene amplification have been most widely used in industry. The popularity of rCHO cells is likely to persist as the demand for therapeutic proteins continues to increase. In this chapter, we review the basic features and the strategies of constructing rCHO cell lines suitable for large-scale production of therapeutic proteins.
Chapter 10
Heterologous Protein Expression in Methylotrophic Bacteria
Kelly A. FitzGerald and Mary E. Lidstrom
Methylotrophic bacteria are attractive hosts for recombinant protein production because of the low cost of the single carbon substrates that sustain them. This cost advantage is particularly notable in the case of labeled substrates for generating labeled proteins. Although a large body of genetic and biochemical work has been performed on the methylotroph Methylobacterium extorquens AM1, the genome has only recently been sequenced and little is known about regulatory mechanisms in this organism. In order to develop strains for commercial production of heterologous proteins, an efficient expression system is necessary. Work in this area is ongoing, and this chapter represents the current state of the field.
Chapter 11
Secretion or Presentation of Recombinant Proteins and Peptides Mediated by the S-layer of Caulobacter crescentus
John F. Nomellini, Michael C. Toporowski, and John Smit
The production of heterologous proteins is a challenge in biotechnology because it is difficult to identify expression systems that are appropriate or capable of expressing proteins of interest. Secretion of heterologous polypeptides is a way to incorporate a significant purification step at the earliest stages of production and there is wide interest in finding bacterial systems capable of doing so in an efficient and faithful manner. The S-layer secretion system of the Gram-negative bacterium Caulobacter crescentus has been adapted for heterologous protein secretion. It is a Type I secretion mechanism that is naturally adapted for high level expression and the C-terminal secretion signal appears to mediate the export of a wide variety of non-native proteins through a large hydrophilic channel that traverses both membranes. The S-layer is a self-assembling, regularly structured surface layer that is present at about 40,000 copies per cell and a candidate for high density display of peptides for a variety of uses ranging from whole cell vaccines to library display. Display in other bacterial systems is often limited by the size of foreign peptides tolerated or the copy number per cell. The S-layer gene of C. crescentus has been adapted for displaying proteins 10-200 amino acids in length at high density and with an unexpectedly high degree of success.
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