title: Vascular endothelial cell antigen (VECA), a novel secreted protein: overexpression in Escherichia coli of the hexahistidine- tagged protein and production of polyclonal antibodies.
abstract: A novel VECA (vascular endothelial cell antigen) was previously identified by using an antibody pool against antigens in HUVECs (human umbilical-vein endothelial cells). VECA has been evolutionarily conserved in vertebrate species ranging from frog and fish to mouse and human. Bioinformatics analysis indicated that VECA was 10.1 kDa in size, with a predicted signal sequence and transmembrane domain, indicating that VECA may have important biological functions. The present paper describes a procedure for obtaining and purifying human recombinant VECA expressed in Escherichia coli as a fusion protein, via a human VECA cDNA linked pQE30 expression vector to DNA coding for hexahistidine. The purified protein was used to raise anti-(human VECA) polyclonal antibodies, which were suitable for detecting the presence of VECA in cells, cell-culture supernatant and tissues by immunoblotting and immunohistochemistry. To our knowledge, this is the first study on the protein expression and polyclonal-antibody production for human VECA. In addition, we report for the first time the positive identification of VECA in humans at the protein and subcellular level and provide the first experimental verification that VECA was indeed a secreted protein. The anti-(human VECA) polyclonal antibodies prepared may serve as a useful tool for future biological function studies on VECA.
authors.affiliation: State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China Medical School, Sichuan University, Chengdu, Sichuan, People's Republic of China.
authors.forename: Shao Quan
authors.initials: SQ
authors.lastname: Xiong
authors.forename: Han Shuo
authors.initials: HS
authors.lastname: Yang
authors.forename: Yi Yuan
authors.initials: YY
authors.lastname: Chen
authors.forename: Cheng Jian
authors.initials: CJ
authors.lastname: Zhao
authors.forename: Zhu
authors.initials: Z
authors.lastname: Yuan
authors.forename: Xin Yu
authors.initials: XY
authors.lastname: Zhao
authors.forename: Fei
authors.initials: F
authors.lastname: Yan
authors.forename: Bai Ding
authors.initials: BD
authors.lastname: Wang
authors.forename: Ming Hai
authors.initials: MH
authors.lastname: Tang
authors.forename: Jin Liang
authors.initials: JL
authors.lastname: Yang
authors.forename: Yu Quan
authors.initials: YQ
authors.lastname: Wei
mesh_terms: D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology* / Q000378:metabolism; D000941:Antigens / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000276:immunology / Q000473:pathology; D003001:Cloning, Molecular / Q000379:methods; D042783:Endothelial Cells / Q000276:immunology*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000276:immunology; D021241:Spectrometry, Mass, Electrospray Ionization; D014594:Uterine Neoplasms / Q000276:immunology / Q000473:pathology
chemical_list: D000906:Antibodies; D000941:Antigens; D011506:Proteins; D011993:Recombinant Fusion Proteins