Studies on the Synthesis of Insulin from Natural and Synthetic A and B Chains. I. Splitting of Insulin and Isolation of the S-Sulfonated Derivatives of the A and B Chains<sup>*</sup>

Katsoyannis, Panayotis G.; Tometsko, Andrew M.; Zalut, Clyde; Stanley, J.; Trakatellis, Anthony C.

doi:10.1021/bi00861a001

Cited by 80 publications

(61 citation statements)

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“…Porcine and bovine insulin were purchased from Sigma. The S-sulfonated derivatives (SS03-) of their A and B chains were prepared and purified as described (13). %5S-Labeled A(SSO3-) and B(SSO3-) were prepared similarly except that 5 mCi of sodium [s5S]sulfite (69 mCi/mol, New England Nuclear) was substituted for unlabeled…”

mentioning

confidence: 99%

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

Goeddel¹,

Kleid²,

Bolívar³

et al. 1979

Proc. Natl. Acad. Sci. U.S.A.

741

249

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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...

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mentioning

confidence: 99%

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

Goeddel¹,

Kleid²,

Bolívar³

et al. 1979

Proc. Natl. Acad. Sci. U.S.A.

741

249

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“…The products obtained by threefold reprecipitation of B(SSb)2 at pH 5-6 were found to be cross-contaminated to the extent of only 1 and 3 %, respectively, as judged by amino acid analysis (lysine and isoleucine as diagnostic amino acids) ( Table 1, columns 3 and 4). A similar, though somewhat less clear-cut, separation has been carried out with the S-sulpho derivatives (Katsoyannis et al, 1967).…”

Section: Resultsmentioning

confidence: 99%

“…When the S-sulpho derivative obtained by reduction and oxidative sulphitolysis of B(SSb)2 was chromatographed on CM-cellulose as described by Katsoyannis et al (1967Katsoyannis et al ( , 1971a, a large additional peak was eluted immediately before B(SS03H)2 (Fig. lb).…”

Section: Resultsmentioning

confidence: 99%

“…Elution at 33ml/h was carried out with 0.04M-sodium acetate/8M-urea (adjusted to pH 4.0 with acetic acid) (Katsoyannis et al, 1967). The fractions were pooled as indicated in Fig.…”

Section: Separation Of S-2-sulphobenzylated A-and B-chainsmentioning

confidence: 99%

“…25,umol), which had been prepared by direct sulphitolysis of insulin (Katsoyannis et al, 1967). A control experiment in which the S-sulpho derivatives of both chains had been obtained by direct sulphitolysis of insulin was run in parallel.…”

Section: -Sulphobenzyl For Protection Of Thiol Groups Of Proteinsmentioning

confidence: 99%

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2-Sulphobenzyl, a new solubilizing and reversible protecting group for cysteine in proteins. Its scope and limitations

Rüegg¹,

Jarvis²,

Rudinger³

1979

Biochemical Journal

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S-2-Sulphobenzylcysteine and S-2-(sulphomethyl)benzylcysteine are prepared by alkylation of cysteine with omega-toluenesultone and 2,3-benzo-1,4-butanesultone respectively. Owing to the presence of the sulphonic acid group, these protected cysteine derivatives are extremely water-soluble and are stable to acid hydrolysis. The groups can be removed by treatment with sodium in liquid NH3. Reduction with tributylphosphine and simultaneous alkylation of insulin with toluenesultone under mild conditions (pH 8.3, aq. 50% propanol) gives the fully S-substituted derivatives in excellent yield; they can be separated by isoelectric precipitation of the S-sulphobenzylated B-chain. Treatment of the latter with sodium in liquid NH3 led simultaneously to the removal of the protecting groups and to the well-documented cleavage at the threonine-proline bond which can be prevented by addition of sodium amide. When deprotected A-chain was recombined with B-chain, insulin was isolated in the same yield and with the same degree of biological activity as that in the control experiment.

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The Greek contribution to diabetes research

Bartsocas

1999

Diabetes Metab. Res. Rev.

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Interest in diabetes mellitus research has escalated in Greece during the last decade. This may be attributed to the realization that diabetes is becoming a major problem for the Greek population, the effect of the St Vincent Declaration in passing specific government legislation, and the founding of the National Hellenic Center for the Prevention and Treatment of Diabetes and its Complications. Research areas include epidemiology, etiopathogenesis, glucose metabolism, complications, prevention and treatment of the disease. Copyright © 1999 John Wiley & Sons, Ltd.

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Studies on the Synthesis of Insulin from Natural and Synthetic A and B Chains. I. Splitting of Insulin and Isolation of the S-Sulfonated Derivatives of the A and B Chains^*

Cited by 80 publications

References 20 publications

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

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

2-Sulphobenzyl, a new solubilizing and reversible protecting group for cysteine in proteins. Its scope and limitations

The Greek contribution to diabetes research

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Studies on the Synthesis of Insulin from Natural and Synthetic A and B Chains. I. Splitting of Insulin and Isolation of the S-Sulfonated Derivatives of the A and B Chains*

Cited by 80 publications

References 20 publications

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

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

2-Sulphobenzyl, a new solubilizing and reversible protecting group for cysteine in proteins. Its scope and limitations

The Greek contribution to diabetes research

Contact Info

Product

Resources

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Studies on the Synthesis of Insulin from Natural and Synthetic A and B Chains. I. Splitting of Insulin and Isolation of the S-Sulfonated Derivatives of the A and B Chains^*