Development of a novel expression system controllable by glucose and temperature

Abstract

A novel system (pTS-C1) to express recombinant proteins in Escherichia coli was constructed. The expression system inducible in the absence of glucose was constructed using ptsG promoter of E.coli. This promoter showed low expression level in the presence of glucose and higher level in the absence of glucose when it is existed as multi-copy. Since high basal expression level of certain genes may induce harmful effects on host cells, Temperature-sensitive C1 repressor derived from bacteriophage P1 was tried to reduce the basal expression level of the constructed expression system. The C1 binding site was introduced to the ptsG promoter region by site-directed mutagenesis. At the permissive temperature (31¡É), C1 binds to its operator site and prevents transcription, while at the nonpermissive temperature (42¡É), C1 is thermally unstable, thereby allowing transcription to proceed. Taken together, the expression using pTS-C1 was low in the presence of glucose at 31¡É, but getting higher with glucose exhaustion followed by temperature shift to 42 ¡É. The expression system was monitored with EGFP forced to the promoter. The expression of EGFP using pTS-C1 system showed the expected expression pattern. The expression was most optimum when SR754 overexpressiong Mlc was used as a host, glucose concentration was 0.5%, and the cultures were incubated at 31¡É for 2.5 to 3 hr. Through this expression system, recombinant proteins could be produced without supplied inducers such as IPTG.