A novel procedure for the preparation of biologically active recombinant peptides using a cyanylation reaction.
A cysteine specific cleavage reaction was used for the preparation of biologically active peptides from recombinant fusion proteins. The fusion protein through cysteine was prepared by a recombinant DNA technology and then treated with cyanylating reagents such as 2-nitro-5-thiocyanatobenzoic acid (NTCB) and 1-cyano-4-(dimethylamino) pyridinium tetrafluoroborate (DMAP-CN) to release the desired product. As an example, we have selected a glucagon-like peptide 1 (7-37) (termed insulinotropin). We constructed an expression vector for a fusion protein in which insulinotropin and human basic fibroblast growth factor (hbFGF) mutein (abbreviated as CS 23) are connected by cysteine and then expressed it in Escherichia coli cells. The fusion protein, after refolding, was purified by heparin affinity chromatography, since CS23 has a strong affinity for heparin. The affinity-purified fusion protein was treated with NTCB or DMAP-CN to give crude insulinotropin, which was then purified by reversed phase (rp) high-performance liquid chromatography (HPLC). From various criteria such as amino acid analysis, amino acid sequence and the biological activity, the purified material obtained was found to be methionylated insulinotropin (Met-insulinotropin) with full activity. The specificity and simplicity of the present method make it versatile and convenient for the preparation of biologically active peptides.
Koyama
N
N
Production Research Laboratories, Takeda Chemical Industries, Ltd., Osaka, Japan.
Kuriyama
M
M
Amano
N
N
Nishimura
O
O
D000595:Amino Acid Sequence; D001483:Base Sequence; D004926:Escherichia coli / Q000378:metabolism; D005934:Glucagon; D052216:Glucagon-Like Peptide 1; D004763:Glucagon-Like Peptides; D008969:Molecular Sequence Data; D010452:Peptide Biosynthesis; D010446:Peptide Fragments; D010455:Peptides; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*
D010446:Peptide Fragments; D010455:Peptides; D011993:Recombinant Fusion Proteins; D004763:Glucagon-Like Peptides; D052216:Glucagon-Like Peptide 1; D005934:Glucagon; C051322:glucagon-like peptide 1 (7-37)