title: Expression, purification, and structural study of the EC4 domain of E-cadherin.
abstract: The objective of this work was to produce unlabeled and 15N-labeled EC4 domain protein from E-cadherin for studying its structure and binding properties to other EC domains as well as to E-cadherin peptides. The EC4 domain of E-cadherin was expressed in Escherichia coli from the vector pASK-IBA6 and localized in the periplasmic space of E. coli. This protein contains a Streptag sequence at the N-terminus, and thus was purified using a Strep-Tactin affinity column. However, at high concentrations the 15N-labeled EC4 protein showed an unstable conformation. Conditions for stabilizing the conformation of this protein were evaluated using CD spectroscopy. The CD results showed that this protein has high conformational stability in Tris buffer at pH 6.0 in the presence of 10 mM calcium chloride.
authors.affiliation: Department of Molecular Biosciences, The University of Kansas, Lawrence, KS 66045, USA.
authors.forename: Kai
authors.initials: K
authors.lastname: Zheng
authors.forename: Irwan T
authors.initials: IT
authors.lastname: Makagiansar
authors.forename: Mengmeng
authors.initials: M
authors.lastname: Wang
authors.forename: Jeffrey L
authors.initials: JL
authors.lastname: Urbauer
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Kuczera
authors.forename: Teruna J
authors.initials: TJ
authors.lastname: Siahaan
mesh_terms: D015820:Cadherins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D002942:Circular Dichroism; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D019897:Periplasm / Q000737:chemistry; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D015820:Cadherins; D018076:DNA, Complementary; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins