In vitro folding of methionine-arginine human lyspro-proinsulin S-sulfonate-disulfide formation pathways and factors controlling yield.
We investigated the in vitro folding of an oxidized proinsulin (methionine-arginine human lyspro-proinsulin S-sulfonate), using cysteine as a reducing agent at 5°C and high pH (10.5-11). Folding intermediates were detected and characterized by means of matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), reversed-phase chromatography (RPC), size-exclusion chromatography, and gel electrophoresis. The folding kinetics and yield depended on the protein and cysteine concentrations. RPC coupled with MALDI-MS analyses indicated a sequential formation of intermediates with one, two, and three disulfide bonds. The MALDI-MS analysis of Glu-C digested, purified intermediates indicated that an intra-A-chain disulfide bond formed first among A6, A7, and A11. Various non-native intra-A (A20 with A6, A7, or A11), intra-B (between B7 and B19), and inter-A-B disulfide bonds were observed in the intermediates with two disulfide bonds. The intermediates with three disulfide bonds had mainly the non-native intra-A and intra-B bonds. At a cysteine-to-proinsulin-SH ratio of 3.5, all intermediates with the non-native disulfide bonds were converted to properly folded proinsulin via disulfide bond reshuffling, which was the slowest step. Aggregation via the formation of intermolecular disulfide bonds of early intermediates was the major cause of yield loss. At a higher cysteine-to-proinsulin-SH ratio, some intermediates and folded MR-KPB-hPI were reduced to proteins with thiolate anions, which caused unfolding and even more yield loss than what resulted from aggregation of the early intermediates. Reducing protein concentration, while keeping an optimal cysteine-to-protein ratio, can improve folding yield significantly.
School of Chemical Engineering, Purdue University, West Lafayette, IN 47907-2100, USA.
Shuang
S
Chen
Lawrence
L
Adijanto
Nien-Hwa Linda
NH
Wang
D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry*; D002845:Chromatography; D002850:Chromatography, Gel; D004220:Disulfides / Q000737:chemistry*; D004586:Electrophoresis; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007328:Insulin / Q000031:analogs & derivatives* / Q000737:chemistry; D061268:Insulin Lispro; D008715:Methionine / Q000737:chemistry*; D008969:Molecular Sequence Data; D011384:Proinsulin / Q000737:chemistry*; D017510:Protein Folding; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D004220:Disulfides; D007328:Insulin; D061268:Insulin Lispro; D011384:Proinsulin; D001120:Arginine; D008715:Methionine