Investigation of the genes coding for chaperonins in Chlorobium tepidum

Abstract

A subgroup of the universally conserved molecular chaperones, the chaperonins assist other proteins in folding or refolding in an ATP- dependent manner. First discovered in E. coli the best described member is the GroEL (Hsp60) and GroES (Hsp10), which are often found together in an operon. The genes for these chaperonins are commonly regulated positive by an alternative sigma factor, as in E. coli or negatively by the CIRCE/ HrcA feedback regulatory system as in streptomyces. C. tepidum, a green sulphur bacterium, contains an operon consisting of groEL and groES, and a single groES-2 gene. In this work extensive analysis of the genetic sequences, in particular the regulatory upstream region was done. This information was used for structure modelling as well as to design primers for isolation and detection of genes by PCR. The isolated genes were subsequently ligated into a pET 102/ D TOPO® vector and expressed in transformed E. coli BL21 (DE) cells. All genes were expressed with a thioredoxin fusion protein in this work. To detect expression of groES, groEL and groES-2 genes in C. tepidum a Real Time reverse transcriptase PCR were done on RNA extracted from C. tepidum. However, this experiment encountered obstacles and were not finished due to lack of time.