Expression in Escherichia coli of chemically synthesized genes for human insulin.

Abstract

Synthetic genes for human insulin A and B chains were cloned separately in plasmid pBR322. The cloned synthetic genes were then fused to an Escherichia coli #-galactosidase gene to provide efficient transcription and translation and a stable precursor protein. The insulin peptides were cleaved from Pgalactosidase, detected by radioimmunoassay and purified. Complete purification of the A chain and partiaf purification of the B chain were achieved. These products were mixed, reduced, and reoxidized. The presence of insulin was detected by radioimmunoassay.Recently improved methods of DNA chemical synthesis, combined with recombinant DNA technology, permit the design and relatively rapid synthesis of modest-sized genes that can be incorporated into prokaryotic cells for gene expression. The feasibility of this general approach was first demonstrated by the synthesis, and expression in Escherichia col, of a gene for the mammalian peptide somatostatin (1).Following the precursor protein approach used for somatostatin (1), the experimental design for this work was such that the insulin peptide chains would be made in vio as short tails joined by a methionine to the end of ,3-galactosidase. After synthesis, the insulin chains, which contain no methionine, can be cleaved off efficiently by treatment with cyanogen bromide. We deliberately chose to construct two separate bacterial strains, one for each of the two peptide chains of insulin: the 21-amino-acid A chain and the 30-amino-acid B chain. In native insulin, the two chains are held together by two disulfide bonds, and methods have been available for years for joining the chains correctly, in vitro, by air oxidation (2). The efficiency of correct joining has been variable and often low. However, by using S-sulfonated derivatives and an excess of A chain, 50-80% correct joining has been obtained (3).The synthetic plan and chemical synthesis of the DNA fragments coding for the A and B chains of human insulin were described in a previous paper (4) and were summarized in Fig. 1 Enzymes and DNA Preparations. T4 DNA ligase and T4 polynucleotide kinase were purified as described (6). Restriction endonuclease EcoRI was purified by the procedure of Greene et al. (7); HindIII was purified by a method developed by D. Goeddel (unpublished). Restriction endonuclease BamHI was purchased from Bethesda Research (Rockville, MD); E. coli alkaline phosphatase was purchased from Worthington.Plasmids, including pBR322 (8), were isolated by a published procedure (9) with some modifications. The chemical synthesis of the deoxyoligonucleotides (figure 1 of ref. 4) has been described (4). Xplac5 DNA was isolated as described (10).The following reaction buffers were used: kinase buffer, 60 mM Tris-HCl, pH 8/15mM 2-mercaptoethanol/10 mM MgCl2; ligase buffer, 20mM Tris-HCl, pH 7.5/10mM dithiothreitol/10 mM MgCl2; BamHI buffer, 20 mM Tris-HCl, pH 7.5/7 mM MgCl2/2 mM 2-mercaptoethanol; EcoRI-HindIll buffer, BamHI buffer containing 50 mM NaCl; and phosphatase buffer, 50 mM Tris-HCl, pH...

New approaches to therapy and diagnosis of diabetes

“…In another approach DNA corresponding to either the A or B chain was inserted into the/~-galactosidase gene of the plasmid pBR 322 [4,20]. The natural promoter of the/3-galactosidase gene initiated expression of a hybrid protein (fl-galactosidase-insulin A or B chain).…”

“…This technology deals with a number of biochemical techniques for handling DNA which include: (1) cutting DNA at specific sites, (2) inserting DNA into bacteria or mammalian cells so that the cell will replicate the DNA and (3) manipulation of cloned DNA so that the host cell makes the protein for which the DNA codes [1][2][3]. Already a number of hormones including somatostatin, human growth hormone and human insulin [4] have been produced by these new methods. However, there are certain criteria which should be considered before new sources of hormones are generally accepted for treatment of human diseases.…”

From crystallography to structural biology, a century of discoveries

“…The whole area might have stalled prematurely. However, shortly after the first successful expression of recombinant proteins in Escherichia coli, including insulin (Goeddel et al, 1979) and somatostatin (Itakura et al, 1977), protein crystallographers turned to recombinant methods as the means to obtain samples for crystallization. Among the very first proteins crystallized using recombinant samples were insulin (Chance et al, 1981), human leukocyte interferon A (Miller et al, 1981(Miller et al, , 1982, murine interferon β (Matsuda et al, 1986), and eglin C (Grutter et al, 1985), Also, recombinant methods made it feasible both the ad hoc modification of protein sequences and the use of orthologues as variables in the crystallization experiments.…”

Qualitative Process Understanding Tools within Bioprocessing: A Case Study

“…The model process chosen, the production of insulin from E. coli, is a complex process, which can be carried out using two methods [16]. Either the chains could be synthesised separately and mixed, reduced and reoxidised after purification [17]. Alternatively, the bacterial culture produces proinsulin, which then undergoes extensive downstream processing to give biologically active insulin [18].…”