期刊
PROTEIN EXPRESSION AND PURIFICATION
卷 93, 期 -, 页码 54-62出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2013.10.015
关键词
Artificial metalloenzymes; Biotin-streptavidin technology; Pichia pastoris; Imine reductase
类别
资金
- Marie Curie Initial Training Network [FP7-ITN-238531]
- Swiss National Foundation (SNF) [200020-144354, 3100A-116507]
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 pPICZ alpha A in frame with the Saccharomyces cerevisiae alpha-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 similar to 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. (C) 2013 Published by Elsevier Inc.
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