High level expression of cellulomonas fimi β-glucosidase gene in escherichia coli

Abstract

To increase expression efficiency of a Cellulomonas fimi $\beta$ -glucosidase gene in Escherichia coli, the gene previously cloned in our laboratory, was subcloned in expression vectors, pKK223-3, pTTQ 19, pIN IIIAI and pPLc2833 all of which have been known to be strong promoters. Plasmid pKK223-3 and pTTQ 19 contain the hybrid promoter tac. Plasmid pIN IIIAI contain the 1pp/lacUV5 promoter and translation initiation signal of 1pp gene, and plasmid pLc2833 contains lambda pL promoter followed by multicloning sites. The $\beta$ -glucosidase gene source were pCF 18 and pCF 16. Recombinant plasmids were obtained by appropriate gene manipulation and the resultant plasmids were named pKCF 16, pTCF 18, pICF 16 and pLCF 16. E. coli cells containing each recombinant plasmids were induced with IPTG and temperature. E. coli JM 103 (pKCF 16) showed very low $\beta$ -glucosidase activity. In the case of E. coli HB 101 cells transformed with pICF 16 or pTC 18, the activity of $\beta$ -glucosidase was increased 11 folds and 24 folds, respectively, compared with that in the E. coli cells carrying pCF 18. The activity of $\beta$ -glucosidase of E. coli M5248 (pLCF 16) was not higher than that of E. coli JM 83 (pCF 18). However, the production of the enzyme protein itself was high. It was estimated that the protein of $\beta$ -glucosidase occupied about 5-8\% of the total cellular proteins and located in cytoplasmic portion of the cell.