Short synthetic oligodeoxyribonucleotide leader sequences enhance accumulation of human proinsulin synthesized in Escherichia coli.

Abstract

Enhanced accumulation of human proinsulin synthesized in Escherchia coil has been achieved by inserting a short leader of homooligopeptide at the amino end of proinsulin. Out of 20 amino acid oligomers studied, (Ala)6, (Asn)6, (Cys)7, (Gln)7, (His)6, (Ser)6, and (Thr)6 leaders were the most effective, with the yield of proinsulin ranging between 6% and 26% of the total bacterial protein. These constructions were made by inserting a synthetic oligodeoxyribonucleotide duplex, coding for a small homooligopeptide, between a synthetic proinsulin gene and an eight-codon f3galactosidase gene residue in vector pUC8. Cyanogen bromide cleavage of the 102 amino acid fused polypeptide yielded a species identical to authentic proinsulin, as judged by NaDodSO4/PAGE and radioimmunoassay.A major problem in the production of low molecular weight proteins by recombinant DNA (Table 1). In each case, the 25-mers were designed to code for maximal length of the homooligopeptide without any termination codon in either strand. Production of Fused Proinsulin. Bacterial cultures (8.0 ml) were grown in double-strength YT medium containing ampicillin at 37°C. After 2 hr, the culture was induced by the addition ofisopropyl -D-thiogalactoside (final concentration 0.7 mM). After 24 hr the cells were harvested by centrifugation. The cell pellet was suspended in 2 ml of 6 M guanidine hydrochloride (pH 7.0) or 1% NaDodSO4, sonicated, and centrifuged. The 1% NaDodSO4 solution was analyzed by NaDodSO4/15% PAGE (11) followed by staining with Coomassie blue (Fig. 2). The lysate solution was also analyzed for C-peptide activity by radioimmunoassay. 561The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Increased expression, folding and enzyme reaction rate of recombinant human insulin by selecting appropriate leader peptide

“…Therefore, researchers have to screen the fusion tags in order to get the optimum result (Hammarström et al, 2002). Even though we cannot exactly determine which fusion tag have a high expression level and stability in our protein, it is considered that the reason of high expression level of H27R is because the increased stability from (His) 10 tag of leader peptide in E. coli and the tendency to stay at the exterior of the proinsulin molecule because of its polarity (Sung et al, 1986). We made a pPT-Q37Rpi plasmid that is 10 amino acids longer than pPT-H27Rpi and compared its expression level.…”

“…Proinsulin, the precursor of insulin, was also produced as a fusion protein, carrying at its N-terminus a B-chain linked by a methionine residue or other amino acid linker (Guo et al, 1984;Sung et al, 1986). Removal of the N-terminal fused peptide linked by methionine is accomplished by chemical cleavage with cyanogen bromide (Mackin and Choquette, 2003).…”

The purification of eukaryotic polypeptides synthesized in Escherichia coli

“…Effects at the level of transcription, translation or on mRNA stability were suggested. Enhanced expression of proinsulin in E. coli by the N-terminal addition of short homo-oligopeptides was described in a recent report (Sung et al, 1986). Seven of the 20 oligomers studied were particularly effective, but the mechanism by which accumulation was affected was not established.…”

Site-Specific Recombination Directed by Single-Stranded Crossover Linkers: Specific Deletion of the Amino-Terminal Region of the β-Galactosidase Gene in pUC Plasmids

“…PLASMi D pUC8 (Vieira and Messing, 1982), a.common cloning vector, is efficient for gene expression (Sung et al, 1986a). However, with all of its unique restriction sites clustered within a /3-galactosidase (/3-Gal) gene, the expressed product of any cloned DNA would be a polypeptide fused to an amino-terminal /3-Gal sequence (Sung et al, 1986a).…”