Vascular endothelial cell antigen (VECA), a novel secreted protein: overexpression in Escherichia coli of the hexahistidine- tagged protein and production of polyclonal antibodies.
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.
Xiong
Shao Quan
SQ
State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China Medical School, Sichuan University, Chengdu, Sichuan, People's Republic of China.
Yang
Han Shuo
HS
Chen
Yi Yuan
YY
Zhao
Cheng Jian
CJ
Yuan
Zhu
Z
Zhao
Xin Yu
XY
Yan
Fei
F
Wang
Bai Ding
BD
Tang
Ming Hai
MH
Yang
Jin Liang
JL
Wei
Yu Quan
YQ
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
D000906:Antibodies; D000941:Antigens; D011506:Proteins; D011993:Recombinant Fusion Proteins