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High-level secretion of recombinant full-length streptavidin in Pichia pastoris and its application to enantioselective catalysis

Nogueira, Elisa S. and Schleier, Thomas and Dürrenberger, Marc and Ballmer-Hofer, Kurt and Ward, Thomas R. and Jaussi, Rolf. (2014) High-level secretion of recombinant full-length streptavidin in Pichia pastoris and its application to enantioselective catalysis. Protein Expression and Purification, 93. pp. 54-63.

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Official URL: http://edoc.unibas.ch/dok/A6338937

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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 650 mg/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 ∼3.2 molecules 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.
Faculties and Departments:05 Faculty of Science > Departement Chemie > Chemie > Bioanorganische Chemie (Ward)
UniBasel Contributors:Nogueira, Elisa S. and Dürrenberger, Marc and Ballmer-Hofer, Kurt and Ward, Thomas R.
Item Type:Article, refereed
Article Subtype:Research Article
Publisher:Elsevier
ISSN:1096-0279
Note:Publication type according to Uni Basel Research Database: Journal article
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Last Modified:03 May 2017 06:52
Deposited On:06 Feb 2015 09:59

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