title: Expression and purification of the functional ectodomain of human anthrax toxin receptor 2 in Escherichia coli Origami B cells with assistance of bacterial Trigger Factor.
abstract: The ectodomain of anthrax toxin receptor 2 (ANTXR2) is composed of a von Willebrand factor A (VWA) domain that binds to anthrax toxin protective antigen (PA) and a newly defined immunoglobulin-like (Ig) domain, in which the disulfide bonds are required for PA pore formation and for the folding of ANTXR2. While the VWA domain has been well characterized, the structure and function of the whole ectodomain (VWA-Ig) are poorly defined, which is mainly due to the limited production of the soluble recombinant protein of the ectodomain. In the present study, the ANTXR2 ectodomain was fused to the C-terminus of bacterial Trigger Factor (TF), a chaperone that mediates the ribosome-associated, co-translational folding of newly synthesized polypeptides in Escherichia coli. Under the control of a cold shock promoter, the fusion protein was overly expressed as a dominant soluble protein at a low temperature in the oxidative cytoplasm of Origami B cells, where formation of the disulfide bonds is favored. Through a series of chromatography, the ANTXR2 ectodomain was purified into homogeneity. The purified ectodomain is functional in binding to PA and mediating PA pore formation on the liposomal membranes, and the yield is applicable for future biochemical and structural characterization.
authors.affiliation: Department of Biological Sciences, University of Texas at El Paso, 500 W. University Avenue, El Paso, TX 79968-0519, United States.
authors.forename: Pedro
authors.initials: P
authors.lastname: Jacquez
authors.affiliation: Department of Biological Sciences, University of Texas at El Paso, 500 W. University Avenue, El Paso, TX 79968-0519, United States.
authors.forename: Ningjing
authors.initials: N
authors.lastname: Lei
authors.affiliation: Department of Biological Sciences, University of Texas at El Paso, 500 W. University Avenue, El Paso, TX 79968-0519, United States.
authors.forename: David
authors.initials: D
authors.lastname: Weigt
authors.affiliation: Department of Chemistry, University of Texas at El Paso, 500 W. University Avenue, El Paso, TX 79968-0519, United States.
authors.forename: Chuan
authors.initials: C
authors.lastname: Xiao
authors.affiliation: Department of Biological Sciences, University of Texas at El Paso, 500 W. University Avenue, El Paso, TX 79968-0519, United States. Electronic address: jsun@utep.edu.
authors.forename: Jianjun
authors.initials: J
authors.lastname: Sun
mesh_terms: D000595:Amino Acid Sequence; D002853:Chromatography, Liquid; D004220:Disulfides; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008081:Liposomes; D008969:Molecular Sequence Data; D019696:Peptidylprolyl Isomerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018000:Receptors, Peptide / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D012995:Solubility
chemical_list: C447150:ANTXR2 protein, human; D004220:Disulfides; D029968:Escherichia coli Proteins; D008081:Liposomes; D018000:Receptors, Peptide; D011993:Recombinant Fusion Proteins; C403324:trigger factor, E coli; D019696:Peptidylprolyl Isomerase
keywords: Anthrax toxin receptor 2; Disulfide bonds; Ectodomain; Trigger Factor