Biotechnol. Appl. Biochem. (2010) 55, 9–28 (Printed in Great Britain) doi:10.1042/BA20090174 9 REVIEW Sense and nonsense from a systems biology approach to microbial recombinant protein production Yanina R. Sevastsyanovich, Sara N. Alfasi and Jeffrey A. Cole 1 School of Biosciences, University of Birmingham, Birmingham B15 2TT, U.K. The ‘Holy Grail’ of recombinant protein production remains the availability of generic protocols and hosts for the production of even the most difficult target products. The present review provides first an explanation why the shock imposed on bacteria using a standard induction protocol not only arrests growth, but also decreases the number of colony-forming units by several orders of magnitude. Particular emphasis is placed on findings of numerous genome-wide transcriptomic studies that highlight cellular stress, in which the general stress, heat-shock and stringent responses are the underlying basis for the manifestation of the deterioration of cell physiology. We then review common approaches used to solve bottlenecks in protein folding and post-translational modification that result in recombinant protein deposition in cytoplasmic inclusion bodies. Finally, we suggest a generic approach to process design that minimizes stress on the produc- tion host and a strategy for isolating improved hosts. Introduction to the diversity of applications of recombinant proteins Many biopharmaceutical projects require the production of a recombinant protein in a heterologous host. In many cases the protein itself is the end product, for example as a biocatalyst for industrial-scale chemical synthesis, for environmental clean-up, as the active component of a biological detergent, as a component of a kit for rapid diagnosis, or even for repeated administration in human or animal healthcare. In such applications, the price of the end product, and hence the profit that can be realized from its successful manufacture, often depend on the production scale and intensity: cheap end products must be produced on a scale large enough to be financially viable. At the other extreme, high-quality proteins are required for NMR or X-ray crystallographic structure determination to enable rational drug design, or for understanding the biology underlying a process. Quality rather than quantity or production intensity now becomes the over-riding requirement. Whatever the end use of the recombinant protein, the ability to express almost any gene at a controllable level makes bacterial hosts and plasmids attractive vehicles for generating the desired product. Despite the availability of a plethora of expression systems, detailed knowledge of the genome sequences, molecular biology, physiology and biochemistry of a range of production hosts, many proteins remain difficult to produce at the scale or quality required. This review will summarize first what commonly happens when a recombinant protein is synthesized in a heterologous bacterial host. Then strategies traditionally used to solve problems of RPP (recombinant protein production) will be listed before the results of more recent transcriptomic and proteomic approaches are assessed. How the production host responds to RPP will be summarized before we step back and ask some fundamental questions regarding why so much accumulated expertise within the bioprocessing sector has failed to achieve the Holy Grail of RPP: the availability of generic protocols and hosts for the production of even the most difficult target product. This will transfer Key words: bacteria, growth arrest, microbial physiology, recombinant protein production (RPP), systems biology. Abbreviations used: AOX1, alcohol oxidase; Arc, anaerobic respiratory control; Bfr2, multicopy suppressor of sensitivity to brefeldin A, involved in secretion; BiP , binding protein, chaperone of Hsp70 family; Bmh2, 14-3-3 protein isoform, involved in vesicle transport; CheY, response regulator of bacterial chemotaxis; Cup5, vacuolar ATP synthase proteolipid subunit; DTT, dithiothreitol; ER, endoplasmic reticulum; ERAD, ER-associated protein degradation; Ero1, Pdi oxidase, functions in oxidative protein folding in ER; GAP promoter, glyceraldehyde-3-phosphate dehydrogenase promoter; GFP , green fluorescent protein; GRAS, generally recognized as safe; GST, glutathione transferase; Hac1, ‘homologous to ATF/CREB 1’, a basic-leucine zipper transcription factor of the mammalian ATF/CREB family; hSOD, human superoxide dismutase; IbpA, inclusion body protein A; IPTG, isopropyl β-D-thiogalactoside; Kin2, serine/threonine protein kinase; Nuc, nuclease; Pdi, protein disulfide isomerase; RESS, repression under secretion stress; RPP , recombinant protein production; scFv, single-chain variable fragment; Ssa4, cytoplasmic member of the Hsp70 family; Sse1, chaperonin in the Hsp110 subclass of the Hsp70 family, ATPase of the Hsp90 chaperone complex; Sso2, plasma membrane t-SNARE (soluble N-ethylmaleimide attachment protein receptor), syntaxin homologue; UPR, unfolded protein response; VP1, viral protein 1. 1 To whom correspondence should be addressed (email j.a.cole@bham.ac.uk). 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