Production and isolation of recombinant somatomedin C.
High-level production of a growth promoting peptide hormone somatomedin C (insulin-like growth factor I) has been achieved using recombinant DNA techniques in Escherichia coli. We found a new structural protein, designated as LH, to stabilize somatomedin C in vivo, and constructed expression vectors for somatomedin C fusing to LH through a methionine and through a tryptophan, respectively. Each of the fused proteins was produced at approximately 4.5 X 10(5) molecules per single E. coli cell and comprised more than 20% of the total cellular proteins. Somatomedin C was obtained in high yield by limited cyanogen bromide degradation of the methionine-type fused protein, in spite of somatomedin C itself having a Met at the 59th position, followed by renaturation of the resultant reduced peptide. The tryptophan-type fused protein was also converted to the peptide hormone by treating with 3-bromo-2-nitrophenylsulphenyl skatole or N-chlorosuccinimide. The recombinant somatomedin C obtained by these procedures was identical with the native one in amino acid sequence as well as in biological activity of stimulation of DNA synthesis in BALB/c 3T3 cells.
D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide; D004926:Escherichia coli / Q000235:genetics; D005813:Genes, Synthetic; D007202:Indicators and Reagents; D007334:Insulin-Like Growth Factor I / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D007986:Luteinizing Hormone / Q000235:genetics; D010446:Peptide Fragments / Q000032:analysis; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D013002:Somatomedins / Q000096:biosynthesis*
D007202:Indicators and Reagents; D010446:Peptide Fragments; D011994:Recombinant Proteins; D013002:Somatomedins; D007334:Insulin-Like Growth Factor I; D007986:Luteinizing Hormone; D003488:Cyanogen Bromide