Expression, purification, and PC1-mediated processing of human proglucagon, glicentin, and major proglucagon fragment.
To examine the cleavage specificity of different members of the furin/propeptide convertase (PC) family of enzymes, we have selected proglucagon (PG) as a model substrate. PG was selected because it is subject to differential processing in vivo. PG is thought to be cleaved initially at an interdomain site to produce glicentin and the major proglucagon fragment (MPGF). These intermediates are subsequently cleaved, most likely by the convertases PC2 and PC1, respectively. To determine the exact sites within PG that are cleaved by PC1 and PC2, we attempted to produce milligram quantities of human PG, glicentin, and MPGF for use in an in vitro conversion assay. A methionine residue was added to the N-terminus of each protein to initiate translation. Purification was achieved using cation exchange and reversed-phase chromatography, and the integrity of the methionylated proteins was confirmed by both electrospray ionization-mass spectrometry and amino acid analysis. The combined expression and purification scheme is fast, efficient, and results in milligram quantities of > or =95% pure proglucagon, > or =95% pure MPGF, and > or =93% pure glicentin. These prohormones are cleaved by PC1 to produce product peptides consistent with the processing of PG observed in vivo, and should therefore be suitable for further analysis of the post-translational processing of PG.
Bonic
Anela
A
Department of Biomedical Sciences, Creighton University School of Medicine, 2500 California Plaza, Omaha, NE 68178-0405, USA.
Mackin
Robert B
RB
D004591:Electrophoresis, Polyacrylamide Gel; D045683:Furin / Q000378:metabolism*; D053771:Glicentin; D005934:Glucagon / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D004763:Glucagon-Like Peptides; D006801:Humans; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D052336:Proglucagon; D011498:Protein Precursors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D021241:Spectrometry, Mass, Electrospray Ionization
D010446:Peptide Fragments; D011498:Protein Precursors; D052336:Proglucagon; D004763:Glucagon-Like Peptides; D053771:Glicentin; D005934:Glucagon; D045683:Furin