Expression of the SpcN Gene from Streptomyces Flavopersicus in Escherichia Coli and Determination of the Gene Function

ABSTRACT

The gene spcN from Straptomyces flavopersicus cloned and sequenced earlier was further haracterized. E. coli expression vector from the pET system was used for overexpression of the protein and characterization of the gene product. SpcN gene was derived as Ndel-HindIII fragment from the plasmid pJOE2686 where it has been cloned earlier. After few subcloning steps the spcN gene was inserted into pET15b as a Ndel-Xhol fragment. The vector containing the target gene was transformed into the expression host E. coli BL21(pLysS). After the induction of the lac promoter by addition of IPTG the expression of the target gene was assessed by analysis of total cell protein on an SDS-polyacrylamide gel followed by Coomassie blue staining. A 30–36 kDa protein was identified which was further purified using His-Tag System and Thrombin cleavage. An enzymic assay confirmed that the gene product of spcN is an ATP-dependent amynoglycoside phosphotransferase.