期刊
ANALYTICAL BIOCHEMISTRY
卷 351, 期 2, 页码 181-186出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2005.11.029
关键词
islet amyloid polypeptide; IAPP; aggregation; disulfide; oxidation; amylin
资金
- NIGMS NIH HHS [GM54233] Funding Source: Medline
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 method is problematic. We have found that the use of I,l,l,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. (c) 2005 Elsevier Inc. All rights reserved.
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