Mitsuo Yanagi

Two enzymes concerned in peptide hormone α-amidation are synthesized from a single mRNA

Summary By expressing truncated rat pituitary ‘peptidylglycine α-amidating enzyme’ cDNAs in COS-7 cells, we found that the two reactions concerned in peptide carboxyl-terminal amidation, namely the peptidylglycine α-hydroxylation reaction and the peptidyl-hydroxyglycine amidation reaction, were catalyzed by 37-and 53-K proteins, which were derived from the 5′- and 3′-coding sequences, respectively. The full-length cDNA directed the expression of both the 37- and 53-K enzymes, and in the combined presence of the two enzymes the full conversion of a glycine-extended peptide into the amidated product was achieved. These results indicated that two enzymes concerned in peptide hormone α-amidation are generated from a common precursor protein encoded by a single mRNA.

High-level synthesis in Escherichia coli of recombinant human calcitonin: Collagenase cleavage of the fusion protein and peptidylglycine α-amidation

Abstract A synthetic gene encoding glycine-extended human calcitonin (hCT-Gly) was inserted between the tac -promoter and the lacZ gene. A synthetic DNA with collagenase recognition sites was placed just downstream at the hCT-Gly gene. In induced cultures, the E. coli cells harboring the gene produced about 0.8 g of the fusion protein (22 mg of hCT-Gly) per liter of culture. The fusion protein was easily and precisely cleaved with a bacterial collagenase to obtain the hCT-Gly. The hCT-Gly was converted with a peptidylglycine α-amidating enzyme to mature hCT, which had hypocalcemic activity similar to that of authentic hCT. These results demonstrate that expression of the fusion gene containing collagenase recognition sites is an effective method for the high-level production of intact peptide hormones.

Expression of Human Calcitonin in Escherichia coli

Calcitonin, which effects skeletal maintenance, is a 32 amino acid thyroid hormone containing a 1-7 disulfide bridge and a C-terminal amide [1]. The use of plasmids is able to overexpress in E. coli via recombinant DNA and may be an effective means for the mass production of hCT1. However, the production of hCT is directly impossible, because the E. coli does not have α -amidating enzyme. We have performed the following strategies.