Genetic construction and high-level gene expression in Escherichia coli of a precursor of salmon calcitonin I.

Abstract

A precursor of salmon calcitonin I (SCT) was produced in Escherichia coli transformed by recombinant plasmids. A double-stranded DNAcoding for SCT-Gly, preceded by ATGand followed by tandem stop codons, was constructed using a combination of chemical synthesis and enzymatic assembly. The gene for the N-terminal portion of metapyrocatechase (C23O) and its preceding ribosome binding site derived from Pseudomonas putida were fused to the synthetic gene. Introduction of this fragment into E. coli resulted in the synthesis of the fusion protein at high efficiency under the control of the tac promoter. After cleavage of the protein with cyanogen bromide, SCT-Gly was purified to homogeneity by gel filtration and by reverse phase chromatography. The structure of the peptide was confirmed by amino acid composition and sequencing analyses. The hypocalcemic activity of the bacterial product was estimated as about one-seventh of that of SCT.

Catechol 2,3-Dioxygenase-catalyzed Synthesis of Picolinic Acids from Catechols

“…5) When the product of C230 reaction was treated with ammonia, a compound identified as picolinic acid was non-enzymatically formed. The structure of the product of C230 reaction was thus elucidated to be 2-hydroxymuconic e-semialdehyde (HMS).8)…”