CAMARGO, Diana C. Rodrigues, Konstantinos TRIPSIANES, Tobias G. KAPP, Joaquim MENDES, Jasmin SCHUBERT, Burghard CORDES and Bernd REIF. Cloning, expression and purification of the human Islet Amyloid Polypeptide (hIAPP) from Escherichia coli. Protein Expression and Purification. SAN DIEGO: Elsevier, 2015, vol. 106, Feb, p. 49-56. ISSN 1046-5928. Available from: https://dx.doi.org/10.1016/j.pep.2014.10.012.
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Basic information
Original name Cloning, expression and purification of the human Islet Amyloid Polypeptide (hIAPP) from Escherichia coli
Authors CAMARGO, Diana C. Rodrigues (276 Germany), Konstantinos TRIPSIANES (300 Greece, guarantor, belonging to the institution), Tobias G. KAPP (276 Germany), Joaquim MENDES (276 Germany), Jasmin SCHUBERT (276 Germany), Burghard CORDES (276 Germany) and Bernd REIF (276 Germany).
Edition Protein Expression and Purification, SAN DIEGO, Elsevier, 2015, 1046-5928.
Other information
Original language English
Type of outcome Article in a journal
Field of Study Genetics and molecular biology
Country of publisher United States of America
Confidentiality degree is not subject to a state or trade secret
WWW URL
Impact factor Impact factor: 1.407
RIV identification code RIV/00216224:14740/15:00087051
Organization unit Central European Institute of Technology
Doi http://dx.doi.org/10.1016/j.pep.2014.10.012
UT WoS 000346113600007
Keywords in English Amyloid; Diabetes; Recombinant protein; human Islet Amyloid Polypeptide (hIAPP); Escherichia coli; Nuclear magnetic resonance (NMR)
Tags ok, rivok
Tags International impact, Reviewed
Changed by Changed by: Mgr. Eva Špillingová, učo 110713. Changed: 6/4/2016 08:59.
Abstract
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 beta-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. (C) 2014 Elsevier Inc. All rights reserved.
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