[23] Short homopeptide leader sequences enhanced production of human proinsulin in Escherichia coli

Publisher Summary This chapter presents an experiment by designing a small protective cap for the efficient expression of the desired polypeptide. In the experiment, a short duplex oligonucleotide, which encodes a homooligopeptide of hydrophilic and protease-resistant amino acid residues, was fused to the proinsulin gene at the amino terminus. Small foreign proteins, which can be produced in bacteria via recombinant DNA, are often degraded rapidly by the proteolytic enzyme system of the host. A widely used approach is to produce the target protein as fusion product with a host protein, which often constitutes an undesirably large portion of the fused polypeptide. This lowers the yield and complicates the purification of the desired protein. The fused gene was found to be expressed efficiently in Escherichia coli . Structure of the cell wall was distorted because of accumulation of the new polypeptide, which resisted degradation. After removal of the homopeptide leader sequence by cyanogens bromide, the fused polypeptide was converted to proinsulin. The chapter discusses the materials and methods required for the expression of the fused protein-containing proinsulin and cyanogen bromide cleavage of the fused protein to give intact proinsulin.