ISSN:
0006-3592
Keywords:
cellulose binding protein
;
high-density fermentations
;
recombinant protein production
;
affinity purification
;
Chemistry
;
Biochemistry and Biotechnology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
,
Process Engineering, Biotechnology, Nutrition Technology
Notes:
A novel expression vector pTugA, previously constructed in our laboratory, was modified to provide kanamycin resistance (pTugK) and used to direct the synthesis of polypeptides as fusions with the C- or N-terminus of a cellulose binding domain which serves as the affinity tag in a novel secretion-affinity fusion system. Fed-batch fermentation strategies were applied to production in recombinant E. coli TOPP5 of the cellulose binding domain (CBD) from the Cellulomonas fimi cellulase Cex. The pTugK expression vector, which codes for the Cex leader sequence that directs the recombinant protein to the periplasm of E. coli, was shown to remain stable at very high-cell densities. Recombinant cell densities in excess of 90 g (dry cell weight)/L were achieved using media and feed solutions optimized using a 2n factorial design. Optimization of inducer (isophenyl-thio-β-D-galactopyranoside) concentration and the time of induction led to soluble, fully active CBDCex production levels in excess of 8 g/L. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55:854-863, 1997.
Additional Material:
5 Ill.
Type of Medium:
Electronic Resource
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