Making, Cloning, and the Expression of Human Insulin Genes in Bacteria: The Path to Humulin

Abstract

In the mid- to late 1970s, recombinant deoxyribonucleic acid methods for cloning and expressing genes in E. coli were under intense development. The important question had become: Can humans design and chemically synthesize novel genes that function in bacteria? This question was answered in 1978 and in 1979 with the successful expression in E. coli of 2 mammalian hormones, first somatostatin and then human insulin. The successful production of human insulin in bacteria provided, for the first time, a practical, scalable source of human insulin and resulted in the approval, in 1982, of human insulin for the treatment of diabetics. In this short review, I give my personal view of how the making, cloning, and expressing of human insulin genes was accomplished by a team of scientists led by Keiichi Itakura, Herbert W. Boyer, and myself.

Keywords: biotechnology; chemical DNA synthesis; genentech; recombinant DNA.

Graphical Abstract

Figure 1.

Schematic of the generation of somatostatin-directed plasmid employed in the transfection E. coli to produce human somatostatin. The chemically synthesized gene for somatostatin was inserted into the E. coli beta-galactosidase gene (ß-gal) on the plasmid pBR322. In E. coli, this plasmid directs the synthesis of a chimeric protein that can be cleaved in vitro at methionine residues by cyanogen bromide to yield active somatostatin. Abbreviations: DNA, deoxyribonucleic acid; Lac, lactose operon; P, lac promoter; O, lac operator; Som, somatostatin Adapted from Itakura K, et al. Expression of Escherichia coli of a chemically synthesized gene for the hormone somatostatin. Science. 1977;198(4321):1056-1063, with the kind permission of the publisher.

Figure 2.

Schematic of the generation of the insulin-directed plasmid employed to transfect E. coli to produce human insulin chains A and B followed by linking via oxidation. As pictured, 2 E. coli strains were engineered to contain chemically synthesized insulin A or B chain genes inserted into the β-galactosidase gene (β-gal) of a plasmid cloning vector. The bacteria made a fused, chimeric protein—β-galactosidase linked by methionine to an insulin tail. After partial purification, the insulin peptide chain is cleaved off by treatment with cyanogen bromide. After separate purification of the insulin A and B chains, they are joined through air oxidation. Adapted from Riggs AD, Itakura K. Synthetic DNA and medicine. Am J Hum Genet. 1979;31(5):531–538, with the kind permission of the publisher.

Figure 3.

Significant advances resulting from the somatostatin and insulin projects.

Figure 4.

Distribution of primary responsibilities for the somatostatin and insulin projects.

Figure 5.

Scientists involved in the somatostatin project at City of Hope, circa 1977. Pictured from left to right: backrow: Arthur Riggs, Herbert Boyer, Keiichi Itakura, Roberto Crea; front row: Lily Xi, Herbert Heyneker, Francisco Bolivar, Leonore Directo, Tadaki Hirose. Photo kindly provided by the City of Hope National Medical Center, Duarte, California.

Figure 6.

Scientists involved in the insulin project, circa 1978. Pictured from left to right are: K. Itakura, A.D. Riggs, D.V. Goeddel, and R. Crea. Photo kindly provided by the City of Hope National Medical Center, Duarte, California.