Very high-level production and export in Escherichia coli of a cellulose binding domain for use in a generic...

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 2(n) factorial design. Optimization of inducer (isophenyl-thio-beta-D-galactopyranoside) concentration and the time of induction led to soluble, fully active CBD(Cex) production levels in excess of 8 g/L.