Chemical synthesis and cloning of gene for A and B chains of human insulin

Abstract

Total synthesis of human insulin genes, 104 and 80 base pairs long for the insulin A and B chains were designed for the expression in the bacterial systems, respectively. The synthetic DNA fragments were synthesized through the phosphite method on solid support. The enzymatic joinings of the oligonucleotides were employed to give the synthetic DNA duplexes, followed by gene cloning to the N-terminus of the beta-galactosidase gene in bacteriophage M13 mp19 and plasmid pUC19. In order to simplify the characterization of clones and the indentification procedures, a couple of DNA fragments encoding short peptides were inserted at the N-terminus of the genes. The usefulness of such devices in conjunction with the expression level of the insulin genes was discussed. Cloned synthetic DNA fragments were sequenced and known that the sequence and leading frame were correct.