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Engineered aggregation inhibitor fusion for production of highly amyloidogenic human islet amyloid polypeptide

Paper ID Volume ID Publish Year Pages File Format Full-Text
23018 43409 2014 7 PDF Available
Title
Engineered aggregation inhibitor fusion for production of highly amyloidogenic human islet amyloid polypeptide
Abstract

•We present an expression system for an extremely aggregation-prone protein.•Fusion of an aggregation inhibitor permits soluble expression of the target protein.•The aggregation inhibitor was selected by phage display from a β-wrapin library.•The method yields pure, monomeric, aggregation-competent islet amyloid polypeptide.•The method is applicable to other aggregation-prone proteins.

Human islet amyloid polypeptide (IAPP) is the major component of pancreatic amyloid deposits in type 2 diabetes. The structural conversion of IAPP from a monomeric state into amyloid assemblies is the subject of intense research. Recombinant production of IAPP is, however, difficult due to its extreme aggregation propensity. Here we describe a novel strategy for expression of IAPP in Escherichia coli, based on an engineered protein tag, which sequesters IAPP monomers and prevents IAPP aggregation. The IAPP-binding protein HI18 was selected by phage display from a β-wrapin library. Fusion of HI18 to IAPP enabled the soluble expression of the construct. IAPP was cleaved from the fusion construct and purified to homogeneity with a yield of 3 mg of isotopically labeled peptide per liter of culture. In the monomeric state, IAPP was largely disordered as evidenced by far-UV CD and liquid-state NMR spectroscopy but competent to form amyloid fibrils according to atomic force microscopy. These results demonstrate the ability of the engineered β-wrapin HI18 for shielding the hydrophobic sequence of IAPP during expression and purification. Fusion of aggregation-inhibiting β-wrapins is a suitable approach for the recombinant production of aggregation-prone proteins.

Keywords
Islet amyloid polypeptide; Protein aggregation; Recombinant expression; Protein engineering; β-Wrapin
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Engineered aggregation inhibitor fusion for production of highly amyloidogenic human islet amyloid polypeptide
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 191, 10 December 2014, Pages 221–227
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us