Recovery and purification of highly aggregation-prone disulfide-containing peptides: application to islet amyloid polypeptide.
Islet amyloid polypeptide (IAPP) is a 37-residue pancreatic hormone. It is responsible for the formation of islet amyloid in vivo and is very insoluble and aggregation-prone in vitro, particularly at basic pH. The peptide contains a disulfide bridge between residues two and seven and an amidated C terminus. There is no reported expression system for the production of amidated IAPP. The peptide is difficult to synthesize and formation of the disulfide by traditional methods is problematic. We have found that the use of 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) or dimethyl sulfoxide (DMSO) significantly improves disulfide formation and purification of highly aggregation-prone IAPP sequences. The use of these organic solvents increases the solubility of the hydrophobic peptides, avoids the use of aqueous basic solutions, and eliminates the need for continuous stirring during oxidation to form the Cys-2 to Cys-7 disulfide bridge. Elimination of the stirring step and basic solution helps to reduce aggregation and allows for more consistent high-performance liquid chromatography (HPLC) retention times. Formation of the intramolecular disulfide using DMSO was found to be the most effective method for IAPP oxidation, reducing the reaction time from 24 to 5 h. Aggregated IAPP can be resolubilized by HFIP or DMSO and recovered by HPLC with very good yield.
Abedini
Andisheh
A
Department of Chemistry, State University of New York at Stony Brook, Stony Brook, NY 11794-3400, USA. aabedini@ic.sunysb.edu
Singh
Gagandeep
G
Raleigh
Daniel P
DP
D000595:Amino Acid Sequence; D000682:Amyloid / Q000138:chemical synthesis / Q000302:isolation & purification*; D000818:Animals; D004121:Dimethyl Sulfoxide / Q000737:chemistry; D004220:Disulfides / Q000138:chemical synthesis; D006801:Humans; D058228:Islet Amyloid Polypeptide; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D020005:Propanols / Q000737:chemistry; D020836:Protein Structure, Quaternary; D016415:Sequence Alignment; D012995:Solubility; D013552:Swine; D006845:Hydrocarbons, Fluorinated
D000682:Amyloid; D004121:Dimethyl Sulfoxide; D004220:Disulfides; D058228:Islet Amyloid Polypeptide; D020005:Propanols; C001337:hexafluoroisopropanol; D006845:Hydrocarbons, Fluorinated