Improved Purification of Proglucagon and Major Proglucagon Fragment and Their Use in a PCSK1 Cleavage Assay

Abstract

Proglucagon (PG) is the precursor of the polypeptide hormones glucagon, glucagon‐like peptide‐1 and glucagon‐like peptide‐2. Maturation of proglucagon takes place via proteolytic cleavage and proceeds through the intermediates glicentin and major proglucagon fragment (MPGF). My laboratory is interested in examining whether the enzymes PCSK1 and PCSK2 are responsible for this maturation. A three‐step purification scheme for recombinant glicentin was previously reported and used as a template for the purification of recombinant PG and MPGF. However, the solid‐phase extraction performed on the initial bacterial extract had to be modified to prevent precipitation of contaminating proteins. In addition, a desirable peak shape for both target proteins could be obtained using anion‐exchange chromatography under less extreme pH conditions and with a shallower salt gradient than required for glicentin. Final purification of PG and MPGF was performed using reversed‐phase chromatography conditions similar to those used for glicentin. Following purification, PG and MPGF (10 μM) were incubated individually with 300 nM PCSK1 at pH 6.0 for 2 hours at 37°C. Under the conditions used, about 70% of PG was cleaved to generate the intermediates glicentin and MPGF; but only about 5% of MPGF was cleaved, and the amount of product produced has been insufficient to establish the identity of the fragments at this time. These results are similar to our previous results using PCSK2 (50% cleavage of PG and less than 10% cleavage of glicentin). We propose that either there are significant differences in prohormone cleavage site susceptibility, or that PCSK1 and PCSK2 are only able to efficiently cleave PG and that additional enzymes are required to perform further endoproteolytic processing of the intermediates.