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Cell-associated degradation affects the yield of secreted engineered and heterologous proteins in the Bacillus subtilis expression system

Lookup NU author(s): Professor Colin Harwood


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A series of chimeric alpha -amylase genes derived from amyL, which encodes the liquefying alpha -amylase from Bacillus licheniformis, were constructed in vitro using gene splicing techniques. The gene constructs were cloned in Bacillus subtilis, where their ability to direct the synthesis and secretion of active alpha -amylase was determined. Detectable alpha -amylase activity was observed for some, but not all, of the chimeric proteins. Studies on the secretion of wildtype AmyL and its chimeric derivatives revealed that, whilst these proteins were stable in the extracellular milieu, all were subject to some degree of degradation during secretion. The chimeric enzymes were degraded to a greater extent than the native enzyme. These findings suggest that cell-associated proteolysis is a significant problem affecting the use of B. subtilis as host bacterium for the production of heterologous proteins.

Publication metadata

Author(s): Harwood C; Jensen CL; Stephenson K; Jorgensen ST

Publication type: Article

Publication status: Published

Journal: Microbiology

Year: 2000

Volume: 146

Issue: 10

Pages: 2583-2594

ISSN (print): 1350-0872

ISSN (electronic): 1465-2080

Publisher: Society for General Microbiology