Heat-shock and stringent responses have overlapping protease activity in Escherichia coli. Implications for heterologous protein yield.
The cellular response of a heat-shocked controlled chemostat of Escherichia coli JM105 [pSH101] was characterized and compared to that of a similar culture induced by isopropyl-beta-D-thiogalactopyranoside (IPTG). The proteases elicited by the IPTG pulse were previously shown to be upregulated by the stringent stress response and were shown here to be upregulated by heat shock, although to a lesser extent. Owing to the apparent overlap between these responses, a relaxed mutant (rel-, devoid of the stringent response; JM109) was examined for its response to both a chemically imposed stringent response and to IPTG induction in controlled chemostats. There was no significant upregulation of protease activity under either imposed stress. More important, a nine-fold increase of chloramphenicol acetyl-transferase (CAT) activity was found for the IPTG-induced relaxed mutant culture. Additionally, the responses from heat shock and IPTG induction were examined in batch cultures. The culture that was simultaneously IPTG-induced and heat-shocked was observed to have the highest CAT activity as well as the most rapid loss in activity after a maximum. Control experiments indicated that the heat shock did not affect loss of CAT activity; instead, the loss of activity correlated with the amount of CAT synthesized. Furthermore, an increase in CAT expression was found during heat shock. Results indicated that heat shock and, alternatively, the use of stringent response-mutant hosts could both be used to facilitate increased recombinant protein yields in the E. coli expression system.
Department of Chemical Engineering, New Mexico State University, Las Cruces, New Mexico 88003, USA.
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Harcum
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Bentley
D019149:Bioreactors; D001709:Biotechnology; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000235:genetics; D010450:Endopeptidases / Q000378:metabolism*; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D006358:Hot Temperature; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D009154:Mutation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
D011994:Recombinant Proteins; D007544:Isopropyl Thiogalactoside; D015500:Chloramphenicol O-Acetyltransferase; D010450:Endopeptidases