Streamlined procedure for the production of normal and altered versions of recombinant human proinsulin.
A method for the simplified, reproducible production of both normal and altered versions of human proinsulin has been developed. A polyhistidine/proinsulin fusion protein was expressed using a prokaryotic expression system and partially purified by affinity chromatography. Disulfide bonds within the polypeptide were formed prior to removal of the affinity tag. The proinsulin cleaved from the fusion protein was then subjected to a final purification step of semipreparative reversed-phase high-performance liquid chromatography. Integrity of both the normal and mutant proinsulins was confirmed by peptide mapping and mass spectrometry. The different versions of proinsulin will be used to map those residues of the substrate used in cleavage site recognition by members of the furin/PC family of converting enzymes.
Mackin
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RB
Department of Biomedical Sciences, Creighton University School of Medicine, 2500 California Plaza, Omaha, Nebraska, 68178-0405, USA. rmackin@creighton.edu
D019943:Amino Acid Substitution; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004926:Escherichia coli; D006639:Histidine; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010455:Peptides; D011384:Proinsulin / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification
D018076:DNA, Complementary; D010455:Peptides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C033223:polyhistidine; D006639:Histidine; D011384:Proinsulin