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

Bacterial Gene Technology, Novo Nordisk A/S, DK-2880 Bagsværd, Denmark¹
Department of Microbiology and Immunology, The Medical School, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne NE2 4HH, UK²

Author for correspondence: Steen T. Jørgensen. Tel: +45 44 44 88 88. Fax: +45 44 42 73 03. e-mail: stjq{at}novo.dk

A series of chimeric -amylase genes derived from amyL, which encodes the liquefying -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 -amylase was determined. Detectable -amylase activity was observed for some, but not all, of the chimeric proteins. Studies on the secretion of wild-type 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.

Keywords: Bacillus subtilis, chimeric proteins, protein secretion, proteolysis, industrial enzymes

Abbreviations: SOE, splicing by overlap extension

^a Present address: Carlsberg Research Laboratory, Gamle Carlsberg vej 10, DK-2500, Copenhagen, Denmark.

^b Present address: Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 N Torrey Pines Road, La Jolla, CA 92037, USA.

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