title: Cell-associated degradation affects the yield of secreted engineered and heterologous proteins in the Bacillus subtilis expression system.
abstract: 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 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.
authors.affiliation: Bacterial Gene Technology, Novo Nordisk A/S, DK-2880 Bagsværd, Denmark1.
authors.forename: Christina L
authors.initials: CL
authors.lastname: Jensen
authors.affiliation: Department of Microbiology and Immunology, The Medical School, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne NE2 4HH, UK2.
authors.forename: Keith
authors.initials: K
authors.lastname: Stephenson
authors.affiliation: Bacterial Gene Technology, Novo Nordisk A/S, DK-2880 Bagsværd, Denmark1.
authors.forename: Steen T
authors.initials: ST
authors.lastname: Jørgensen
authors.affiliation: Department of Microbiology and Immunology, The Medical School, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne NE2 4HH, UK2.
authors.forename: Colin
authors.initials: C
authors.lastname: Harwood
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D003001:Cloning, Molecular; D005818:Genetic Engineering / Q000379:methods; D007091:Image Processing, Computer-Assisted / Q000379:methods; D015151:Immunoblotting / Q000379:methods; D010957:Plasmids / Q000235:genetics; D011233:Precipitin Tests; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D014169:Transformation, Bacterial; D000516:alpha-Amylases / Q000235:genetics* / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D000516:alpha-Amylases