Expression, purification, and PC1-mediated processing of (H10D, P28K, and K29P)-human proinsulin.
Our previous methods for the generation of recombinant human proinsulin were inadequate in terms of reproducibility and yield. In addition, it was difficult to perform structure/function studies on proinsulin because of its tendency to form hexamers. We have developed an improved procedure, which overcomes many of the technical purification problems, and results in a potentially monomeric version of modified proinsulin. Inclusion bodies were prepared using a commercial bacterial lysis solution. The inclusion bodies were solubilized and the fusion protein's affinity tag was removed by chemical cleavage. The polypeptide was then reduced and transferred into a refolding buffer. Following an overnight incubation, only a single form of proinsulin was detected using analytical reversed-phase high-performance liquid chromatography. The refolded (H10D, P28K, and K29P)-human proinsulin (DKP-hPI) was subjected to a final purification step using reversed-phase chromatography. The method is reproducible and produces milligram quantities of purified DKP-hPI from a single liter of bacterial culture. The final product is greater than 95% pure and is suitable for use as a substrate for the propeptide convertase PC1.
Mackin
Robert B
RB
Department of Biomedical Sciences, Creighton University School of Medicine, 2500 California Plaza, Omaha, NE 68178-0405, USA. rmackin@creighton.edu
Choquette
Meredith H
MH
D000595:Amino Acid Sequence; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000378:metabolism; D004220:Disulfides; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011384:Proinsulin / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification*; D043484:Proprotein Convertases; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D013329:Structure-Activity Relationship; D013997:Time Factors
D004220:Disulfides; D010455:Peptides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D004247:DNA; D011384:Proinsulin; D043484:Proprotein Convertases; D016282:Aspartic Acid Endopeptidases