COMPARISON OF TEMPERATURE-INDUCED AND ISOPROPYL-BETA-D-THIOGALACTO-PYRANOSIDE-INDUCED SYNTHESIS OF BASIC FIBROBLAST GROWTH-FACTOR IN HIGH-CELL-DENSITY CULTURES OF RECOMBINANT ESCHERICHIA-COLI

Two different expression systems were developed for expression of the cDNA encoding human basic fibroblast growth factor (bFGF) using Escher ichia coli TG1 as host organism. The bFGF structural gene was cloned i nto two vectors differing only with respect to the promoter, which was either the bacteriophage lambda PRPL- or the E. coli lac-promoter. Th e resulting expression systems were studied in high-cell density cultu res. Cells were grown in a fed-batch procedure with a predetermined ex ponential feeding rate based on mass balances and kinetic equations to ensure constant specific growth rates. Prior to induction, cells were grown at 30 degrees C. Product formation was induced by either a temp erature shift from 30 to 42 degrees C or by the addition of isopropyl- beta-D-thiogalactopyranoside (IPTG). Under comparable culture conditio ns-induction of bFGF expression at 41 to 45 g l(-1) dry cell weight an d the addition of feed medium with identical rates-bFGF accumulated to 4.9 and 1.1 g l(-1) using the temperature- and IPTG-inducible express ion systems, respectively. The final biomass concentrations obtained u sing temperature- and IPTG-inducible expression systems were 61 and 13 5 g l(-1) dry cell weight, whereas the specific product concentrations were 80 and 8.1 mg bFGF g(-1) dry cell weight, respectively. When a t emperature shift was used for product induction, 30% of bFGF was recov ered as inclusion bodies in the insoluble cell fraction. IPTG-dependen t induction yielded exclusively soluble bFGF.