Prokaryotic expression, purification, and production of polyclonal antibody against human polypeptide N-acetylgalactosaminyltransferase 14
- 作者：Chen Wu ; YuanYuan Wang ; MinJi Zou ; YaoJun Shan ; GuangYin Yao ; Ping Wei ; GuangYu Chen ; JiaXi Wang ; DongGang Xu
- 关键词：GalNAc-T14 ; Prokaryotic expression ; Purification ; Polyclonal antibody
- 刊名：Protein Expression and Purification
- 出版年：2007
- 期刊代码：184_10465928
- 类别：bio
- 出版时间：November 2007
- 卷：56
- 期：1
- 页码：1-7
- 文件大小：527 K
摘要
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-β-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.