title: Localized production of human E-cadherin-derived first repeat in Escherichia coli.
abstract: E-cadherin is a cell surface adhesion molecule that is expressed in both epithelial and endothelial tissues. In this study, an improved method for the simple production of the human E-cadherin-derived first repeat E-CAD1 was developed by exporting it into the periplasmic space of Escherichia coli. Localization of the recombinant protein into the periplasm allowed the isolation of E-CAD1 without cell lysis. The N-terminus of E-CAD1 is fused to a streptavidin-derived peptide to allow single-step purification using a Streptag affinity column. Optimal expression in LB medium produced 3.2 mg/L while expression in minimal medium containing 15NH(4)Cl as the sole source of nitrogen produced 4.2 mg/L purified (15)N-labeled E-CAD1. Heteronuclear NMR spectroscopy confirmed that the purified E-CAD1 produced in this manner was correctly folded. The expression and purification protocol for unlabeled and isotopically labeled E-CAD1 permits rapid preparative production of this protein for mechanistic and structural studies.
authors.affiliation: Department of Pharmaceutical Chemistry, University of Kansas, 2095 Constant Avenue, Lawrence, KS 66047-2504, USA.
authors.forename: Irwan T
authors.initials: IT
authors.lastname: Makagiansar
authors.forename: Atsutoshi
authors.initials: A
authors.lastname: Ikesue
authors.forename: Phuong Duc
authors.initials: PD
authors.lastname: Nguyen
authors.forename: Jeffrey L
authors.initials: JL
authors.lastname: Urbauer
authors.forename: Ramona J Bieber
authors.initials: RJ
authors.lastname: Urbauer
authors.forename: Teruna J
authors.initials: TJ
authors.lastname: Siahaan
mesh_terms: D015820:Cadherins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D010446:Peptide Fragments / Q000737:chemistry; D019897:Periplasm / Q000378:metabolism; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D015820:Cadherins; D003470:Culture Media; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins