Expression, purification and characterization of recombinant human proinsulin

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 chromato...

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Bibliographic Details
Published in:FEBS letters 1997-02, Vol.402 (2), p.124-130
Main Authors: Cowley, Darrin J, Mackin, Robert B
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Chromatography, Ion Exchange
Cloning, Molecular
Expression
Gene Library
Humans
Molecular Sequence Data
Peptide Fragments - chemistry
Peptide Fragments - isolation & purification
Peptide Mapping
Plasmids
Polymerase Chain Reaction
Proinsulin
Proinsulin - biosynthesis
Proinsulin - chemistry
Proinsulin - isolation & purification
Protein Conformation
Protein Folding
Recombinant
Recombinant Fusion Proteins - biosynthesis
Recombinant Proteins - biosynthesis
Recombinant Proteins - chemistry
Recombinant Proteins - isolation & purification
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Summary: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.
ISSN:0014-5793
1873-3468
DOI:10.1016/S0014-5793(96)01511-6