Chemical synthesis of a human interferon-α<sup>2</sup>, gene and its expression in<i>Escherichia coli</i>

Edge, Michael D.; Greene, A.R.; Heathcliffe, G.R.; Moore, Valerie; Faulkner, N.J.; Camble, Roger; Petter, N.N.; Trueman, P.; Schuch, Wolfgang; Hennam, J.; Atkinson, T; Newton, Clive R.; Markham, Alexander F.

doi:10.1093/nar/11.18.6419

Cited by 35 publications

(10 citation statements)

References 38 publications

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“…Synthesis of the Gene. The approach adopted for preparing the gene consisted of synthesizing oligodeoxynucleotide segments representing the desired sequence for the two DNA strands and ligating them to form the complete gene (18)(19)(20)(21)(22)(23). The designed fragment carrying the CSa gene was divided into three subfragments ("thirds") extending from nucleotide residues 1 to 60, 61 to 157, and 158 to 253.…”

Section: Resultsmentioning

confidence: 99%

“…The ability to test additional variant or mutant forms of C5a would provide insight concerning which residues are important for its biological activity. Recent advances in recombinant DNA technology now make it feasible both to approach the problem of inadequate supply of a scarce protein and to generate a system for mutagenesis by cloning the gene coding for the protein and having it expressed in bacteria (18)(19)(20)(21)(22)(23)(24). Chemical synthesis is a preferred method to obtain a C5a gene, since C5a is a cleavage product of a larger protein.…”

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confidence: 99%

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Chemical synthesis of a gene encoding the human complement fragment C5a and its expression in Escherichia coli.

Mandecki¹,

Mollison²,

Bölling³

et al. 1985

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

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A gene coding for the C5a fragment of the fifth component of human complement has been chemically synthesized, cloned, and expressed in Escherichia cofl. The 253-base-pair gene fragment was built through a two-step enzymic assembly of 16 oligonucleotides, the average length of each being 32 residues. The oligonucleotides were synthesized by using the phosphoramidite method. The gene was cloned in a pBR322-derivative plasmid downstream from the lac uppromoter mutant, UVS-D. The expression of C5a was detected and measured by immunoassay and a radioligand binding assay. C5a from E. coli was comparable to C5a2purified from human serum in inhibiting binding of human 1 I-labeled C5a to its putative receptor on polymorphonuclear leukocytes. Studies of smooth muscle contraction in isolated guinea pig ileum showed that the recombinant C5a was biologically active and produced cross-tachyphylaxis with human serum-derived C5a. The results demonstrate the feasibility of expressing C5a anaphylatoxin in bacteria and provide a system for mutagenesis of the C5a protein.Activation of the complement system generates several biologically active peptides. One of these, C5a, is a potent stimulant of inflammatory processes and potentially plays an important role in the pathogenesis of a number of inflammatory diseases (1, 2). C5a is a glycoprotein ofMr 11,000 (3). During serum complement activation, CSa is cleaved from the amino end of the a chain of the fifth component of complement, C5, by a trypsin-like enzyme, C5 convertase (4). Once CSa is liberated, the carboxyl-terminal arginine is rapidly removed by serum carboxypeptidase N, forming the desarginine derivative, des-Arg-C5a (5). CSa is a potent stimulator of a variety of neutrophil functions thought to be involved in inflammation, including chemotaxis, chemokinesis, aggregation, lysosomal enzyme release, and the generation of toxic oxygen products (6-10). In addition, C5a produces smooth muscle contraction and vasodilation (11,12), promotes release of histamine from basophils and mast cells (13,14), and stimulates antibody synthesis (15). Des-Arg-C5a is capable of producing many of these effects but is less potent than C5a (7,12).Although the sequence of its 74 amino acids has been elucidated (3), efforts to determine the secondary and tertiary structure of C5a and to relate these features to the function of the molecule have been limited by difficulties in obtaining suitable quantities of C5a. In a typical isolation, <0.5 mg of pure C5a can be recovered from 1 liter of complement-activated serum (16). For this reason, there is a paucity of sequence-homology information, as only one other mammalian C5a (porcine) has been sequenced (17). The ability to test additional variant or mutant forms of C5a would provide insight concerning which residues are important for its biological activity. Recent advances in recombinant DNA technology now make it feasible both to approach the problem of inadequate supply of a scarce protein and to generate a system for mutagenesis by clonin...

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Chemical synthesis of a gene encoding the human complement fragment C5a and its expression in Escherichia coli.

Mandecki¹,

Mollison²,

Bölling³

et al. 1985

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

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“…Enzymatic ligation of the 78 chemically synthesized oligonucleotides was carried out by joining [5][6][7][8][9][10] pieces in the first step. The subfragments thus obtained were further ligated to yield 415-bp (A+B segment: amino-terminus portion) and 176-bp (C segment: carboxylterminus portion) polynucleotides.…”

Section: Resultsmentioning

confidence: 99%

“…Deoxyoligonucleotides with a chain length of [7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26] bases were synthesized by the phosphotriester solid-phase method, using 1% cross-linked polystyrene (15) as the support. Altogether 78 chains were prepared by addition of dinucleotide blocks to the nucleosides linked through a 3'-succinyl group to the resin.…”

Section: Methodsmentioning

confidence: 99%

Synthesis of a gene for human growth hormone and its expression in Escherichia coli.

Ikehara¹,

Ohtsuka²,

Tokunaga³

et al. 1984

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

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A gene coding for human growth hormone, which consists of 192 amino acids, was chemically synthesized.The synthesis entailed ligating 78 deoxyribooligonucleotides, which had been synthesized on polymer supports by the phosphotriester method with frequently occurring amino acid codons of Escherichia coli. The chemically synthesized gene was inserted into an E. coli plasmid downstream from the E. coli thp promoter, with a modified ribosome-binding region carried on pBR322. E. coli cells transformed with this recombinant plasmid synthesized 2.9 x 106 molecules per cell of human growth hormone upon induction. The induced polypeptide was identical with natural human growth hormone in size and in immunological properties, as well as in biological activity as examined by the tibial test with hypophysectomized rats.The improvement of techniques in chemical synthesis of deoxyribooligonucleotides has made possible the total synthesis of genes of 100-500 nucleotides (1-9). By this technique, genes with designed amino acid sequences can in principle be synthesized, and the products of their expression in bacteria can be examined. In the present work, a gene for human growth hormone (hGH) (10), which contains 191 amino acids and methionine, was totally synthesized by chemical means, using amino acid codons most frequently appearing in Escherichia coli (11-13) (Fig. 1). To establish a general procedure for efficient expression of genes for relatively large peptides, hGH was thought to be a suitable target. Use of the synthetic gene will provide sufficient sites for restriction enzymes, which should be useful in mutations of genes for replacement of protein domains. Rapid synthesis of 78 gene fragments of 7-26 bases was carried out, followed by sequential ligation to construct the gene of 584 base pairs (bp). This is the longest gene so far synthesized chemically. Separately, a modified trp promoter was constructed that allows expression of any synthetic genes with an initiation codon placed downstream of it. A recombinant plasmid carrying the promoter and the hGH gene produced the hormone in E. coli at a high level. The induced polypeptide was indistinguishable from the natural hGH in several features. The purified hormone was as active as the natural growth hormone in biological tests (14). MATERIALS AND METHODSDeoxyoligonucleotides. Deoxyoligonucleotides with a chain length of 7-26 bases were synthesized by the phosphotriester solid-phase method, using 1% cross-linked polystyrene (15) as the support. Altogether 78 chains were prepared by addition of dinucleotide blocks to the nucleosides linked through a 3'-succinyl group to the resin. Tetranucleotide blocks prepared by condensation of dimers in the liquid phase were used for the synthesis of the 25-mer (A-UO; Fig. 1). Sixteen dinucleotide blocks were prepared with mesitylenesulfonyl-3-nitro-triazolide (16) by methods essentially as reported (17) and purified mostly by reversed-phase chromatography (18) under conditions described (19,20). A typical synthesis of a 1...

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“…The therapeutic efficacy of human IFNs has been established in the treatment of certain human cancers and viral diseases (1). Recombinant human IFN-␣ (rh-IFN-␣) is used for the treatment of hairy cell leukemia, AIDS-related Kaposi's sarcoma, and chronic hepatitis B and C (2,3) and has been obtained by the expression of the IFN-␣ gene in Escherichia coli (4) or yeast (5).…”

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confidence: 99%

Affinity Purification of Recombinant Interferon-α on a Mimetic Ligand Adsorbent

Swaminathan

Khanna

1999

Protein Expression and Purification

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Chemical synthesis of a human interferon-α², gene and its expression inEscherichia coli

Cited by 35 publications

References 38 publications

Chemical synthesis of a gene encoding the human complement fragment C5a and its expression in Escherichia coli.

Chemical synthesis of a gene encoding the human complement fragment C5a and its expression in Escherichia coli.

Synthesis of a gene for human growth hormone and its expression in Escherichia coli.

Affinity Purification of Recombinant Interferon-α on a Mimetic Ligand Adsorbent

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Chemical synthesis of a human interferon-α2, gene and its expression inEscherichia coli

Cited by 35 publications

References 38 publications

Chemical synthesis of a gene encoding the human complement fragment C5a and its expression in Escherichia coli.

Chemical synthesis of a gene encoding the human complement fragment C5a and its expression in Escherichia coli.

Synthesis of a gene for human growth hormone and its expression in Escherichia coli.

Affinity Purification of Recombinant Interferon-α on a Mimetic Ligand Adsorbent

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Chemical synthesis of a human interferon-α², gene and its expression inEscherichia coli