Cloning and Overexpression Studies on the Glutathione Peroxidase-Like Protein from the Cyanobacterium Nostoc punctiforme ATCC 29133

Abstract

Cyanobacteria are oxygen-evolving photosynthetic prokaryotes possessing a wide range of enzymatic antioxidants to tackle oxidative stress if and when it arises in cells. Glutathione peroxidase is an important enzyme in the antioxidative machinery of cells and may confer protection to various stress conditions. In the diazotrophic cyanobacterium Nostoc punctiforme ATCC 29133 (hereafter N.punctiforme), an open reading frame (ORF) encoding for a putative glutathione peroxidase was identified as NpunR_4660 by bioinformatics approach. This 486 bp ORF was amplified from the genomic DNA of N.punctiforme by polymerase chain reaction. The forward and reverse primers used for cloning the ORF contained NdeI and XhoI at their 5’ and 3’ end, respectively. The PCR amplified product was sub-cloned into pJET cloning vector. Further the ORF was introduced into pET-20B vector for overexpression of the corresponding protein in Escherichia coli BL21 DE3 strain. After induction of E.coli cultures by Isopropylthiogalactoside (IPTG) and, Sodium dodecyl sulphate–polyacrylamide gel electrophoresis of cell-free extracts, an approximately 18 kDa protein was detected in the cytoplasm, which matched with the theoretical molecular weight of the protein. This protein was not present in control cultures transformed with pET-20B without the cyanobacterial ORF. These results suggest that foreign enzymes such as glutathione peroxidase can be produced in large amounts for biotechnological purposes successfully even in cells of a different origin like E.coli.