1. Vascular endothelial cell antigen (VECA), a novel secreted protein: overexpression in Escherichia coli of the hexahistidine- tagged protein and production of polyclonal antibodies
- Author
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Jin Liang Yang, Fei Yan, Bai Ding Wang, Yi Yuan Chen, Shao Quan Xiong, Cheng Jian Zhao, Ming Hai Tang, Zhu Yuan, Han Shuo Yang, Xinyu Zhao, and Yu Quan Wei
- Subjects
Signal peptide ,Spectrometry, Mass, Electrospray Ionization ,Recombinant Fusion Proteins ,Blotting, Western ,Molecular Sequence Data ,Biomedical Engineering ,Bioengineering ,Breast Neoplasms ,Biology ,Applied Microbiology and Biotechnology ,Antibodies ,law.invention ,law ,Complementary DNA ,Drug Discovery ,Escherichia coli ,Humans ,Amino Acid Sequence ,Antigens ,Cloning, Molecular ,Peptide sequence ,Expression vector ,Process Chemistry and Technology ,Endothelial Cells ,Proteins ,General Medicine ,Molecular biology ,Fusion protein ,Immunohistochemistry ,Protein Structure, Tertiary ,Transmembrane domain ,Polyclonal antibodies ,Uterine Neoplasms ,biology.protein ,Recombinant DNA ,Molecular Medicine ,Female ,Biotechnology - 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.
- Published
- 2008