title: Expression, purification and characterization of recombinant human proinsulin.
abstract: We have recently developed a method to produce native human proinsulin using a bacterial expression system. A proinsulin fusion protein was recovered from inclusion bodies and cleaved using cyanogen bromide. The released proinsulin polypeptide was S-sulfonated and purified by anion exchange chromatography. Following refolding, proinsulin was purified by reversed-phase high-performance liquid chromatography. Combined peptide mapping and mass spectrometric analysis indicated that the proinsulin contained the correct disulfide bridging pattern. This proinsulin will be used to study the specificity of the furin/PC family of converting enzymes by using it as a substrate in a recently developed assay.
authors.affiliation: Department of Biomedical Sciences, Creighton University School of Medicine, Omaha, NE 68178, USA.
authors.forename: D J
authors.initials: DJ
authors.lastname: Cowley
authors.forename: R B
authors.initials: RB
authors.lastname: Mackin
mesh_terms: D000595:Amino Acid Sequence; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D015723:Gene Library; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010449:Peptide Mapping; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011384:Proinsulin / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D011487:Protein Conformation; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D016324:Sequence Tagged Sites
chemical_list: D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D011384:Proinsulin