Efficient production of extracellular proteins with Escherichia coli by means of optimized coexpression of bacteriocin release proteins.
Aiming to facilitate the accessibility of recombinant proteins produced with Escherichia coli, extracellular expression may be achieved by means of bacteriocin release protein (BRP) coexpression. Different types of BRPs were tested in order to optimize protein secretion into the culture medium. Those included the well-studied BRPs of the Colicin E1 and Cloacin DF13 bacteriocins and variants thereof. BRP expression was stringently controlled by means of the arabinose inducible P(BAD) promoter, which accounts for a broad-range adjustment of expression strength. Using appropriate arabinose concentrations, a concentration range was determined, that allowed efficient secretion of the model proteins alkaline phosphatase and beta-lactamase, with 90-95% of the proteins released into the culture medium. Kinetic investigations into BRP expression and protein secretion revealed a rapid increase of extracellular protein concentration within 5-10 min past induction. Alternatively to fine-tuned BRP expression during cultivation, protein production and secretion could be decoupled by establishment of appropriate induction strategies and up to 90% of alkaline phosphatase was released into the culture medium within 3h after reaching maximum biomasss concentrations. Both, fine-tuned and growth decoupled BRP expression accounted for extracellular alkaline phosphatase concentrations of roughly 500 mg l(-1) of culture broth and selectivities of 50mg of this enzyme per gram of cell dry mass, respectively.
Sommer
Benjamin
B
Department of Fermentation Engineering, Faculty of Technology, Bielefeld University, POB 100 131, D-33501 Bielefeld, Germany.
Friehs
Karl
K
Flaschel
Erwin
E
D000469:Alkaline Phosphatase / Q000378:metabolism; D001430:Bacteriocins / Q000378:metabolism*; D018533:Biomass; D001709:Biotechnology / Q000379:methods*; D003470:Culture Media; D004926:Escherichia coli / Q000254:growth & development / Q000378:metabolism*; D005110:Extracellular Space / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics; D007700:Kinetics; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000378:metabolism; D001618:beta-Lactamases / Q000378:metabolism
D001430:Bacteriocins; D003470:Culture Media; D011994:Recombinant Proteins; D000469:Alkaline Phosphatase; D001618:beta-Lactamases