An approach to high-level production of a mecasermin (somatomedin C) fused protein in Escherichia coli HB101

Abstract A method to produce a mecasermin fused protein on an industrial scale was investigated. We constructed a new expression vector (designated pLSD1) derived from pLHSdMmtrp (Saito, Y. et al. : J. Biochem., 101, 123–134, 1987) by introduction of a synthetic fd phage terminator and deletion of the rop region originating from pBR322. The plasmid, pLSD1, exhibited high stability and was present at high copy number in Escherichia coli HB101. Although the growth of E. coli HB101 pLSD1 was not sufficient for high-level production of the fused protein in a Trp-deficient medium such as M9CA medium, it was improved by growing the strain in a medium containing 0.7% yeast extract which was constantly supplied with glucose. E. coli HB101 requires Pro and Leu for its growth; however, excess Leu tended to inhibit the cell growth. From the results of investigation of the mechanism of inhibition by Leu, addition of Ile was found to prevent the inhibition. Consequently, high-level production of the fused protein was attained by (i) using pLSD1 as the expression vector, (ii) cultivation of E. coli HB101 pLSD1 in a medium containing 0.7% yeast extract, (iii) fed-batch cultivation with periodic addition of glucose, and (iv) addition of Pro, Leu and Ile during cultivation. The cell density reached an OD at 600 nm of 50.4 and production of the fused protein was 1.24 g/ l broth in a 150 l fermentor.