Use of folding modulators to improve heterologous protein production in Escherichia coli

Abstract

Despite the fundamental importance of E. coli in the manufacture of a wide range of biotechnological and biomedical products, extensive process and/or target optimisation is routinely required in order to achieve functional yields in excess of low mg/l levels. Molecular chaperones and folding catalysts appear to present a panacea for problems of heterologous protein folding in the organism, due largely to their broad substrate range compared with, e.g., protein-specific mutagenesis approaches. Painstaking investigation of chaperone overproduction has, however, met with mixed -and largely unpredictable -results to date. The past 5 years have nevertheless seen an explosion in interest in exploiting the native folding modulators of E. coli, and particularly cocktails thereof, driven largely by the availability of plasmid systems that facilitate simultaneous, non-rational screening of multiple chaperones during recombinant protein expression. As interest in using E. coli to produce recombinant membrane proteins and even glycoproteins grows, approaches to reduce aggregation, delay host cell lysis and optimise expression of difficult-toexpress recombinant proteins will become even more critical over the coming years. In this review, we critically evaluate the performance of molecular chaperones and folding catalysts native to E. coli in improving functional production of heterologous proteins in the bacterium and we discuss how they might best be exploited to provide increased amounts of correctly-folded, active protein for biochemical and biophysical studies.

Production of prone‐to‐aggregate proteins

“…When lacking specific knowledge of suitable chaperones, screens for expression in the presence of combinations of chaperones can be conducted [55]. A comprehensive review examining the advantages and disadvantages of this approach can be found in a review by Kolaj et al [56].…”

Secretion of Anti-Plasmodium Effector Proteins from a Natural Pantoea agglomerans Isolate by Using PelB and HlyA Secretion Signals

“…Proteins are folded into their final conformation, and the PelB leader is removed in the periplasm (5). Problems in the periplasm may have been encountered, such as periplasmic inclusion bodies, errors in folding or disulfide bond formation, or degradation (24,46). Any of these problems could explain why SM1 was detected in the cell pellet and not in the supernatant and why the anti-Pbs21 scFv was not detected in P. agglomerans.…”

Rehosting of Bacterial Chaperones for High-Quality Protein Production

“…In the widely used bacterium Escherichia coli, many strategies have been explored to enhance recombinant protein solubility. Among them, the coproduction of folding modulators, believed to be limiting in cells actively producing recombinant proteins, has attracted special attention (14). However, the efficacy of such an approach has been highly controversial.…”

“…In contrast to the widespread use of folding modulators in bacteria (14), very few attempts have been made to improve cytoplasmic protein production in insect cells with the aid of We appreciate financial support through grants BIO2007-61194 and EUI2008-03610 (MICINN) to A.V. and grant BB/C504735/1 (BBSRC) to P.R.…”