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

Abstract

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...

Synthesis of a gene for the protein kinase domain of the epidermal growth factor receptor and its expression in Escherichia coli

“…1, by silent mutagenesis of the DNA sequence. Amino acid codons were chosen to maximise expression in E. coli as discussed previously [7,81. Sequences of more than six identical residues were identified using a microcomputer and altered to avoid mismatches in duplex alignment.…”

“…A plasmid expression vector (originally constructed for expression of synthetic human growth hormone genes [7]) containing the synthetic c-Ha-ras gene was used for cloning of the EGF-receptor protein kinase domain gene. This vector, derived from plasmid pBR322, carries the trp promoter of E. coii and the synthetic ribosome binding region of the trp promoter upstream of the synthetic c-Ha-ras gene (Fig.…”

“…The chemically synthesised gene (0.5 pg) was then mixed with 1.25 pg of the Sun-CluI fragment of pRR61 in standard ligation buffer containing 10 mM 2-mercaptoethanol and incubated with T4 DNA ligase for 2 h at 20°C. Calcium-chloride-treated E. coli HBlOl cells were treated with the ligation reaction mixture (dissolved in 10 p1 Tris/EDTA pH 8.0) and plated on ampicillin-containing agar plates using essentially the same procedure as described previously [7,81. After selection of clones carrying the synthetic gene, the base sequence was confirmed by standard dideoxy-sequencing procedures with commercially available kits using 7-deazaGTP [ 101 and dITP.…”

Fed-batch culture of Escherichia coli harboring a runaway-replication plasmid.

“…Amounts and periods of the additions were determined from previous ex- Expression of the ampicillin resistance gene on the plasmid was examined by the assay of /Mactamase activity.17) Cells collected by centrifugation were washed with potassium phosphate buffer (0.1 mol//, pH 7.0), suspended in the same buffer and sonicated by using a cell disrupter (Ohtake Works Co., Type 5202).…”

Synthesis and hybridization of dodecadeoxyribonucleotides containing a fluorescent pyridopyrimidine deoxynucleoside

“…The 3'-phosphorylated nucleosides (4) and dinucleotides containing the major nucleosides were prepared as previously described (3). Dinucleotides containing the analog (5) were synthesized using nucleotide (3).…”