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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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A T4 lysozyme-coding DNA sequence of 495 bp was chemically synthesized and cloned by ligation of 26 deoxyribooligonucleotide fragments in two steps with a linearized plasmid followed by transformation. On selection by colony hybridization and DNA sequence analysis, clone pTLY.10 was identified to contain a complete T4 lysozyme synthetic DNA. On expression under lac-promoter, unfused T4 lysozyme was obtained in approximately 4-6% yield. The design and synthesis of two putative folding mutants, flexible (Gly-Gly-Gly) and rigid (Asn-Asp-Gly) at position 73-74-75, were based on hierarchical principles. Both mutants lost enzymatic activity of the wildtype. These results are readily understandable if the hierarchical organization of the structure is taken into account. A possible explanation is that the catalytic sites are blocked in both mutants.