THERMAL INDUCTION EFFECTS ON HTLV-I ENVELOPE PROTEIN SYNTHESIS IN RECOMBINANT ESCHERICHIA COLI

Abstract

The effects of induction temperature, induction holding time and temperature cycling range on cell growth and rp21E expression in Escherichia coli K12 MZl pKS400 were studied with the overall aim of optimizing the production of human T-cell lymphotropic virus type-I (HTLV-I) envelope protein (p21E) . Temperature effect on cycling range was found to have a significant final cell density. Significant increase in maximum rp21E concentration was achieved using multiple-cycle step induction between 36° and 39°C and holding times of 30 and 60 minutes as a consequence of the higher final cell density attained. The effects of varying the induction holding times during temperature cycling was less marked. Whereas a holding time of zero (saw-tooth profile) was detrimental for protein expression, the differences in OD and expression levels between holding times of 30 and 60 minutes were minimal. A comparison of sustained temperature induction at 42° and 39°C revealed virtually similar profiles of cell density and protein expression level. Our results indicate that, in a fed-batch fermentation with temperature induction, cell viability is not solely affected by expression of the recombinant protein, but rather by the combined effects of the metabolic stress caused by exposure at elevated temperature and temperature cycling. A comparison of the acetic acid concentration profile for all the various induction strategies shows that acetic acid assimilation was inhibited at higher temperatures, in the range 36° to 39°C. Two major heat shock proteins, GroEL and DnaK, were observed to be overexpressed after temperature shift to 39°C.