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Open Access Green as soon as Postprint is submitted to ZB.
Cloning, expression and purification of the human Islet Amyloid Polypeptide (hIAPP) from Escherichia coli.
Protein Expr. Purif. 106, 49-56 (2015)
Type II diabetes is characterized by deposition of the hormone human Islet Amyloid Polypeptide (hIAPP). Formation of hIAPP amyloid fibrils and aggregates is considered to be responsible for pancreatic β-cell losses. Therefore, insight into the structure of hIAPP in the solid-state and in solution is of fundamental importance in order to better understand the action of small molecules, which can potentially dissolve protein aggregates and modulate cell toxicity. So far, no procedure has been described that allows to obtain the native human IAPP peptide at high yields. We present here a cloning, expression and purification protocol that permits the production of 2.5 and 3 mg of native peptide per liter of minimal and LB medium, respectively. In the construct, hIAPP is fused to a chitin binding domain (CBD). The CBD is subsequently cleaved off making use of intein splicing reaction which yield amidation of the C-terminus. The N-terminus contains a solubilization domain which is cleaved by V8 protease, avoiding additional residues at the N-terminus. The correct formation of the disulfide bond is achieved by oxidation with H2O2.
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Publication type
Article: Journal article
Document type
Scientific Article
Keywords
Amyloid ; Diabetes ; Escherichia Coli ; Nuclear Magnetic Resonance (nmr) ; Recombinant Protein Human Islet Amyloid Polypeptide (hiapp)
ISSN (print) / ISBN
1046-5928
e-ISSN
1046-5928
Quellenangaben
Volume: 106,
Pages: 49-56
Publisher
Elsevier
Non-patent literature
Publications
Reviewing status
Peer reviewed
Institute(s)
Institute of Structural Biology (STB)