Prokaryotic expression, purification, and production of polyclonal antibody against human polypeptide N-acetylgalactosaminyltransferase 14.
Polypeptide N-acetylgalactosaminyltransferase 14 (GalNAc-T14, EC 2.4.1.41) belongs to a large subfamily of glycosyltransferases residing in the Golgi apparatus. N-Acetylgalactosaminyltransferases (GalNAc-Tases) catalyze the first step in the O-glycosylation of mammalian proteins by transferring N-acetyl-D-galactosamine (GalNAc) to peptide substrates. Here, the cloning, expression, purification, and polyclonal antibody preparation of GalNAc-T14 were described. A full-length GalNAc-T14 cDNA was inserted in a prokaryotic expression plasmid pGEX-4T-1 at the EcoRI and XhoI restriction sites. pGEX-4T-T14 was highly expressed in Escherichia coli (E. coli) BL21(DE3) cells after induced by isopropyl-beta-D-thiogalactoside (IPTG). The expressed GST-GalNAc-T14 fusion protein was purified by GSTrap FF chromatography and then used as antigen to immunize rabbits. The obtained antiserum was precipitated by 50% saturated ammonium sulfate and then purified by DEAE-Sepharose FF chromatography. To confirm the activity and specificity of the GalNAc-T14 antibody, we constructed the plasmid pFLAG-GalNAc-T14 to transfect transiently HEK 293T cells. Transiently expressed FLAG-GalNAc-T14 was identified by Western blot analysis with GalNAc-T14 antibody and FLAG monoclonal antibody, respectively. The production of the polyclonal antibody against GalNAc-T14 provides a good tool for studying the biofunctions of GalNAc-T14.
Wu
Chen
C
Laboratory of Molecular Genetics, Beijing Institute of Basic Medical Sciences, 27 Taiping Road, Beijing 100850, China.
Wang
YuanYuan
Y
Zou
MinJi
M
Shan
YaoJun
Y
Yao
GuangYin
G
Wei
Ping
P
Chen
GuangYu
G
Wang
JiaXi
J
Xu
DongGang
D
D000818:Animals; D015153:Blotting, Western; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D017350:N-Acetylgalactosaminyltransferases / Q000276:immunology*; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000302:isolation & purification; D000097763:Polypeptide N-acetylgalactosaminyltransferase
D017350:N-Acetylgalactosaminyltransferases; D011993:Recombinant Fusion Proteins; D000097763:Polypeptide N-acetylgalactosaminyltransferase; C471038:UDP-N-acetyl-D-galactosamine polypeptide N-acetylgalactosaminyltransferase 14, human