title: Constitutive production and efficient secretion of soluble full-length streptavidin by an Escherichia coli 'leaky mutant'.
abstract: Due to its various applications the protein streptavidin is a highly interesting target for heterologous production. This study focuses on different Escherichia coli-based constructs targeting a high-level expression and secretion of streptavidin to the medium. The effect of various promoters, variants of the target gene, leader sequences and host strains on expression and secretion into the culture broth was analyzed. Constitutive production of full-length streptavidin fused with the leader sequence of the bglA gene from Bacillus amyloliquefaciens by the periplasmic 'leaky mutant' E. coli JW1667-5 (Δlpp-752:kan) at 30°C generated the highest yield of the conditions tested, surpassing the extracellular concentration of a conventional T7-based expression system. Supplementation of the medium by the non-ionic surfactants Triton(®) X-100 and X-45 led to an improved secretion of the protein to the culture supernatant. Tetrameric concentrations of streptavidin of 2790±166nM were reached in shake flasks at a productivity of 49.6nMh(-1). Optimization of conditions led to a successful transfer to the bioreactor, yielding a maximal concentration of 2608±169nM and a productivity of 65.2nMh(-1) in fed-batch operation. The proportion of biotin-blocked binding sites of 8.3±4.3% indicated a highly bioactive product.
authors.affiliation: Lehrstuhl für Fermentationstechnik, Technische Fakultät, Universität Bielefeld, PF 10 01 31, D-33501 Bielefeld, Germany. Electronic address: jmu@fermtech.techfak.uni-bielefeld.de.
authors.forename: Jakob Michael
authors.initials: JM
authors.lastname: Müller
authors.affiliation: Lehrstuhl für Fermentationstechnik, Technische Fakultät, Universität Bielefeld, PF 10 01 31, D-33501 Bielefeld, Germany.
authors.forename: David
authors.initials: D
authors.lastname: Wetzel
authors.affiliation: Lehrstuhl für Fermentationstechnik, Technische Fakultät, Universität Bielefeld, PF 10 01 31, D-33501 Bielefeld, Germany.
authors.forename: Erwin
authors.initials: E
authors.lastname: Flaschel
authors.affiliation: Lehrstuhl für Fermentationstechnik, Technische Fakultät, Universität Bielefeld, PF 10 01 31, D-33501 Bielefeld, Germany.
authors.forename: Karl
authors.initials: K
authors.lastname: Friehs
authors.affiliation: Lehrstuhl für Fermentationstechnik, Technische Fakultät, Universität Bielefeld, PF 10 01 31, D-33501 Bielefeld, Germany.
authors.forename: Joe Max
authors.initials: JM
authors.lastname: Risse
mesh_terms: D020121:5' Untranslated Regions; D001407:Bacillus / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D061249:Batch Cell Culture Techniques; D003001:Cloning, Molecular; D003470:Culture Media / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development*; D009154:Mutation; D011401:Promoter Regions, Genetic; D019809:Streptavidin / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism
chemical_list: D020121:5' Untranslated Regions; D001426:Bacterial Proteins; D003470:Culture Media; D019809:Streptavidin
keywords: Chain length of streptavidin; Escherichia coli; Fed-batch cultivation; Periplasmic leaky mutant; Secretion; Temperature