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 responses as indicated by an increase in ATP-dependent proteolysis are tested. A set of experiments was carried out where glucose, IPTG (inducer), and phenylalanine (rate-limiting precursor) were added gradually in a fed-batch fashion. A significant increase in CAT activity was found compared with pulse-induction. In addition, there was a substantial increase in the rate of CAT synthesis as well as in the final specific CAT activity when phenylatanine and the inducer were added simultaneously. CAT degradation was confirmed through Western blotting analysis. Protease analysis (SDS-GPAGE) indicated lower proteolytic activity for the IPTG and phenylalanine fed-batch cases. GroEL immunoas-says indicated that amplification of stress proteins occurred upon CAT induction. This research impacts the yield of soluble cytoplasmic proteins in Escherichia coli and suggests that metabolically based induction/feeding policies are beneficial. (c) 1995 John Wiley & Sons Inc.
Ramírez
D M
DM
Center for Agricultural Biotechnology and Department of Chemical Engineering, University of Maryland, College Park, Maryland 20742.
Bentley
W E
WE