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Proteomic analysis of Bacillus subtilis strains engineered for improved production of heterologous proteins

Lookup NU author(s): Dr Susanne Pohl, John Hulme, Dr Goksel Misirli, Dr David Radford, Dr Wayne Smith, Professor Anil Wipat, Professor Colin Harwood

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Abstract

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.


Publication metadata

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

Journal: Proteomics

Year: 2013

Volume: 13

Issue: 22

Pages: 3298-3308

Print publication date: 01/11/2013

ISSN (print): 1615-9853

ISSN (electronic): 1615-9861

Publisher: Wiley-Blackwell Publishing Ltd.

URL: http://dx.doi.org/10.1002/pmic.201300183

DOI: 10.1002/pmic.201300183


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