Expression, purification and characterization of recombinant human proinsulin

Darrin J. Cowley; Robert B. Mackin

doi:10.1016/S0014-5793(96)01511-6

Expression, purification and characterization of recombinant human proinsulin

Darrin J. Cowley, Robert B. Mackin

Department of Biomedical Sciences

Research output: Contribution to journal › Article › peer-review

56 Scopus citations

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.

Original language	English (US)
Pages (from-to)	124-130
Number of pages	7
Journal	FEBS Letters
Volume	402
Issue number	2-3
DOIs	https://doi.org/10.1016/S0014-5793(96)01511-6
State	Published - Jan 27 1997

All Science Journal Classification (ASJC) codes

Biophysics
Structural Biology
Biochemistry
Molecular Biology
Genetics
Cell Biology

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10.1016/S0014-5793(96)01511-6

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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.",

author = "Cowley, {Darrin J.} and Mackin, {Robert B.}",

note = "Funding Information: The authors would like to thank Eli Lilly and Co. for providing us with human proinsulin, Meredith Choquette for technical assistance and her help with manuscript preparation, and Robert Allington for help with the expression and purification of the recombinant proinsulin. This work was funded by grants from the State of Nebraska Cancer and Smoking-related Disease Program and the Health Future Foundation.",

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AU - Cowley, Darrin J.

AU - Mackin, Robert B.

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N2 - 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.

AB - 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.

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Expression, purification and characterization of recombinant human proinsulin

Abstract

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