Expression of the Streptomyces griseus α-amylase gene in Escherichia coli

Abstract

The amy gene of Streptomyces griseus was not expressed in Escherichia coli cells due to the lack of recognition of the amy promoter by the E. coli RNA polymerase, as confirmed by using promoter-probe vectors. The expression of the amy gene in E. coli was detected only when the promoter-less gene was placed under the control of the lacZ promoter and was dependent on the level of IPTG added to the medium. The extracellular alpha-amylase detected in the culture broth seems to be released by cellular lysis. When the amy gene lacking both leader peptide and promoter was transcribed from the lacZ promoter, no alpha-amylase activity was detected but larger E. coli cells and inclusion bodies were observed.

Biochemical characterization of the SecA protein of Streptomyces lividans

“…A 2.3-kb EcoRIϪBglII fragment, containing the S. griseus amy gene [26], was subcloned into EcoRIϪBamHI digested pGEM-3zf(ϩ), giving rise to the recombinant plasmid pGEM3z-amy. This plasmid was mutated by site-directed mutagenesis (using the Quikchange Site-Directed Mutagenesis Kit; Stratagene) to generate a NdeI restriction site at the amy translation-initiation start codon.…”