Preparation of Tyr-C-peptide from genetically altered human insulin precursor.
C-peptide radioimmunoassay (C-peptide RIA) is widely used in determination of pancreatic B-cell secretion activity. 125I labeled Tyr-C-peptide is indispensable in C-peptide RIA kit. Herein we discuss a way of obtaining recombinant Tyr-C-peptide. Arg32Tyr human pro-insulin mutant (R32Y-proinsulin) gene was constructed by site-directed mutagenesis and overexpressed in Escherichia coli. Purified R32Y-proinsulin was converted to insulin and Tyr-C-peptide by trypsin and carboxypeptidase B codigestion. Tyr-C-peptide was isolated through reverse-phase HPLC (RP-HPLC) and identified by C-peptide RIA and amino acid analysis.
Sun
H
H
Department of Biochemistry and Molecular Biology, College of Life Sciences, Peking University, Beijing, China.
Tang
J
J
Hu
M
M
D001483:Base Sequence; D002096:C-Peptide / Q000302:isolation & purification / Q000378:metabolism*; D043424:Carboxypeptidase B; D002268:Carboxypeptidases / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D017931:DNA Primers / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011384:Proinsulin / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D011863:Radioimmunoassay; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D015183:Restriction Mapping; D014357:Trypsin / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism
D002096:C-Peptide; D017931:DNA Primers; D011994:Recombinant Proteins; D014443:Tyrosine; D011384:Proinsulin; D002268:Carboxypeptidases; D043424:Carboxypeptidase B; D014357:Trypsin