FED-BATCH FEEDING AND INDUCTION POLICIES THAT IMPROVE FOREIGN PROTEIN-SYNTHESIS AND STABILITY BY AVOIDING STRESS RESPONSES

In this work, feeding policies aimed to avoid cellular stress response s as indicated by an increase in ATP-dependent proteolysis are tested. A set of experiments was carried out where glucose, IPTG (inducer), a nd phenylalanine (rate-limiting precursor) were added gradually in a f ed-batch fashion. A significant increase in CAT activity was found com pared with pulse-induction. In addition, there was a substantial incre ase in the rate of CAT synthesis as well as in the final specific CAT activity when phenylalanine and the inducer were added simultaneously. CAT degradation was confirmed through Western blotting analysis. Prot ease analysis (SDS-GPAGE) indicated lower proteolytic activity for the IPTG and phenylalanine fed-batch cases. GroEL immunoassays indicated that amplification of stress proteins occurred upon CAT induction. Thi s research impacts the yield of soluble cytoplasmic proteins in Escher ichia coli and suggests that metabolically based induction/feeding pol icies are beneficial. (C) 1995 John Wiley and Sons, Inc.