title: High-level secretion of recombinant full-length streptavidin in Pichia pastoris and its application to enantioselective catalysis.
abstract: Artificial metalloenzymes result from the incorporation of a catalytically competent biotinylated organometallic moiety into full-length (i.e. mature) streptavidin. With large-scale industrial biotechnology applications in mind, large quantities of recombinant streptavidin are required. Herein we report our efforts to produce wild-type mature and biotin-free streptavidin using the yeast Pichia pastoris expression system. The streptavidin gene was inserted into the expression vector pPICZαA in frame with the Saccharomyces cerevisiae α-mating factor secretion signal. In a fed-batch fermentation using a minimal medium supplemented with trace amounts of biotin, functional streptavidin was secreted at approximately 650mg/L of culture supernatant. This yield is approximately threefold higher than that from Escherichia coli, and although the overall expression process takes longer (ten days vs. two days), the downstream processing is simplified by eliminating denaturing/refolding steps. The purified streptavidin bound ∼3.2molecules of biotin per tetramer. Upon incorporation of a biotinylated piano-stool catalyst, the secreted streptavidin displayed identical properties to streptavidin produced in E. coli by showing activity as artificial imine reductase.
authors.affiliation: Department of Chemistry, University of Basel, Spitalstrasse 51, CH-4056 Basel, Switzerland.
authors.forename: Elisa S
authors.initials: ES
authors.lastname: Nogueira
authors.forename: Thomas
authors.initials: T
authors.lastname: Schleier
authors.forename: Marc
authors.initials: M
authors.lastname: Dürrenberger
authors.forename: Kurt
authors.initials: K
authors.lastname: Ballmer-Hofer
authors.forename: Thomas R
authors.initials: TR
authors.lastname: Ward
authors.forename: Rolf
authors.initials: R
authors.lastname: Jaussi
keywords: Artificial metalloenzymes; Biotin–streptavidin technology; Imine reductase; Pichia pastoris