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Recombinant analogues of human parathyroid hormone [hPTH-(1-84)] were expressed in Escherichia coli harboring plasmids containing synthetic genes under the control of the lac promoter. The level of expression of the gene encoding the truncated analogue, hPTH-(3-84), was greater than that of the gene encoding full-length hPTH-(1-84) but less than that of the gene encoding proparathyroid hormone (hProPTH). This may be due in part to the relative efficiency of translation of the mRNA as suggested by secondary structure analysis and in part because of enhanced stability of the extended peptide. Formylmethionyl derivatives of hProPTH and of hPTH-(3-84) and underivatized hPTH-(3-84) were purified by HPLC, and their identity was confirmed by NH2-terminal sequencing and amino acid analysis. The bioactivity of these recombinant peptides was then tested in skeletal and renal adenylate cyclase assays in vitro and in assays examining effects on plasma and urine calcium and phosphate levels and on urine cyclic AMP levels in vivo. The NH2-terminally extended analogue fMet-hProPTH displayed 10% of the in vitro activity of hPTH-(1-84) and was a partial agonist in vivo. The peptides hPTH-(3-84) and fMet-hPTH-(3-84) were inert in vitro and were very weak in vitro antagonists when compared to the NH2-terminal analogue bovine [Nle8,18Tyr34]PTH-(3-34)-NH2. In vivo, hPTH-(3-84) and the bPTH-(3-34) analogue, when assayed at a 10:1 molar ratio relative to bPTH-(1-84), were each inert, and neither demonstrated antagonist activity at these concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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The conversion of D-xylose to ethanol by the yeast Pachysolen tannophilus is relatively inefficient in batch culture. The inefficiency has been attributed in part to concurrent utilization of ethanol in the presence of appreciable concentrations of D-xylose and to the formation of xylitol and other by-products. To increase the concentration of ethanol accumulated in batch cultures, UV-induced mutants of P. tannophilus were selected on the basis of diminished growth on ethanol. Eleven independent mutant loci that conferred the ethanoldefective phenotype were identified. Three led to a greater yield and volumetric rate of production of ethanol than the wild type. (ne also produced less xylitol and was characterized by a deficiency in activity for malate dehydrogenase.