Purification of the des‐(31,32) and des‐(64,65) intermediates of proinsulin conversion

Abstract

The biosynthesis of insulin requires proteolytic processing of a larger precursor, proinsulin (PI), by three different proteases: proprotein convertase 1 (PC1), PC2 and carboxypeptidase E. The conversion process must proceed through one of two intermediates, des‐(31,32)‐PI or des‐(64,65)‐PI, to reach the final product, insulin. I am interested in examining the structure of the intermediates to help explain the specificity of the enzyme‐substrate interactions involved in PI conversion. I have previously reported the use of partial trypsin/carboxypeptidase B digestion to generate the intermediates, and the use of anion‐exchange chromatography to separate the intermediates from the substrate and products using anion‐exchange chromatography. The intermediates have now been separated from one another and purified from miscleaved PI using reversed‐phase high performance liquid chromatography. Starting from 5 mg of PI, 0.4 mg of des‐(31,32)‐PI at 99% purity and 0.2 mg of des‐(64,65)‐PI at 90% purity can be obtained. It should therefore be possible to scale up purification of the intermediates for structural characterization and examination of PI cleavage specificity.