[Design of a hybrid gene coding for the leader sequence of Bacillus amyloliquefaciens alpha-amylase and for human proinsulin].

Abstract

The chemically synthesized structure gene of human proinsulin was cloned in E. coli on the secretory vector containing regulatory elements of the Bacillus amyloliquefaciens alpha-amylase gene. The proinsulin gene was inserted by the EcoRI site located immediately after the DNA area encoding the alpha-amylase signal peptide. The E. coli cells transformed by such a plasmid produced hybrid protein consisting of the alpha-amylase signal peptide, five amino acid residues after the gene mating and human proinsulin. For accurate mating of the alpha-amylase gene leader sequence and proinsulin gene directed mutagenesis was performed on the filiform phage M13 mp9 with synthetic oligonucleotide. The hybrid gene was transferred to the vector molecule capable of replicating in Bacillus subtilis. It was shown that in the cells of both E. coli and B. subtilis there is synthesized protein interacting by the radio-immunological data with antibodies to porcine insulin, a large portion of immunologically active protein being detected in the periplasmic space of E. coli cells and in the culture fluid of B. subtilis cells which was indicative of proinsulin secretion directed by the alpha-amilase regulatory elements.