Chloramphenicol acetyltransferase activity and GroEL

Abstract

Several proteins were overproduced by the powerful T7 expression system, and their solubility and enzyme activity were detemined. Also effects of a chaperone GroEL on chloramphenicol acetyltransferase (CAT) assembly and $\beta$-lactamase solubility were investigated in this study. Using promoter specificity and strong transcriptional activity of phage T7 RNA polymerase, selective overproductions of CAT, $\beta$-lactamase, GroEL and GroES were accomplished at high levels in E. coli BL21(DE3) which carries the IPTG inducible T7 RNA polymerase gene in the chromosome. CAT, GroES and GroEL were completely soluble, and overproduced CAT was fully active, Precursor forms of $\beta$-lactamase in cytoplasm were mostly insoluble and mature forms in periplasm were mostly soluble. Next, interaction between GroEL and CAT, or GroEL and $\beta$-lactamase was investigated. In the CAT overproducing strain with wild type GroEL, defects of CAT activity was not observed. Overproduction of GroE proteins either by a heat shock or from multiple copies of the gene did not increase CAT specific activity. Also two different mutations of GroEL did not have an effect. In-all cases the CAT specific activity was shown olny as much as it was produced. Thus, GroEL does not seem to be critical in vivo in the assembly and active structure formation of CAT, although they have been previously shown to interact with each other in vitro. Although GroEL interacts with $\beta$-lactamase both in vitro and in vivo, overproduced GroEL proteins did not change insoluble precursor forms of $\beta$-lactamase in the cytoplasm, probably because a molar ratio of GroE over $\beta$-lactamase was apparently below 1. Finally, some applications of the T7 expression system were tried. The hybrid T7/SP6 RNA polymerase appeared upon IPTG induction in a soluble form. However, the hybrid RNA polymerase failed to transcribe cat gene under either T7 or SP6 promoter. Expression of cat under E. coli promoter was inhibited by...