Lookup NU author(s): Dr Susanne Pohl,
Dr Goksel Misirli,
Dr David Radford,
Dr Wayne Smith,
Professor Anil Wipat,
Professor Colin Harwood
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The use of bacterial systems for recombinant protein production has advantages of simplicity, time and cost over competing systems. However, widely used bacterial expression systems (e.g. Escherichia coli, Pseudomonas fluorescens) are not able to secrete soluble proteins directly into the culture medium. This limits yields and increases downstream processing time and costs. In contrast, Bacillus spp. secrete native enzymes directly into the culture medium at grams-per-litre quantities, although the yields of some recombinant proteins are severely limited. We have engineered the Bacillus subtilis genome to generate novel strains with precise deletions in the genes encoding ten extracytoplasmic proteases that affect recombinant protein secretion, which lack chromosomal antibiotic resistance genes. The deletion sites and presence of single nucleotide polymorphisms were confirmed by sequencing. The strains are stable and were used in industrial-scale fermenters for the production of the Bacillus anthracis vaccine protein, protective antigen, the productivity of which is extremely low in the unmodified strain. We also show that the deletion of so-called quality control proteases appears to influence cell-wall synthesis, resulting in the induction of the cell-wall stress regulon that encodes another quality control protease.
Author(s): Hulme J; Wipat A; Pohl S; Harwood CR; Smith W; Radford DS; Misirli G; Bhavsar G; Bloor AE; Leckenby MW; Williamson ED; Cranenburgh RM
Publication type: Article
Publication status: Published
Print publication date: 01/11/2013
ISSN (print): 1615-9853
ISSN (electronic): 1615-9861
Publisher: Wiley-Blackwell Publishing Ltd.
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