Kurkela Mika, García-Horsman J Arturo, Luukkanen Leena, Mörsky Saila, Taskinen Jyrki, Baumann Marc, Kostiainen Risto, Hirvonen Jouni, Finel Moshe
Viikki Drug Discovery Technology Center, University of Helsinki, Finland.
J Biol Chem. 2003 Feb 7;278(6):3536-44. doi: 10.1074/jbc.M206136200. Epub 2002 Nov 14.
Eight human liver UDP-glucuronosyltransferases (UGTs) were expressed in baculovirus-infected insect cells as fusion proteins carrying a short C-terminal extension that ends with 6 histidine residues (His tag). The activity of recombinant UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT2B4, UGT2B7, and UGT2B15 was almost fully inhibited by 0.2% Triton X-100. In the case of UGT1A9, however, glucuronidation of alpha-naphthol and scopoletin was resistant to such inhibition, whereas glucuronidation of entacapone and several other aglycones was sensitive. His-tagged UGT1A9 was purified by immobilized metal-chelating chromatography (IMAC). Purified UGT1A9 glucuronidated scopoletin at a high rate, whereas its glucuronidation activity toward entacapone was low and largely dependent on phospholipid addition. Recombinant UGT1A9 in which the His tag was replaced by hemagglutinin antigenic peptide (HA tag) was also prepared. Insect cells were co-infected with baculoviruses encoding both HA-tagged and His-tagged UGT1A9. Membranes from the co-infected cells, or a mixture of membranes from separately infected cells, were subjected to detergent extraction and IMAC, and the resulting fractions were analyzed for the presence of each type of UGT1A9 using tag-specific antibodies. In the case of separate infection, the HA-tagged UGT1A9 did not bind to the column. When co-infected with His-tagged UGT1A9, however, part of the HA-tagged enzyme was bound to the column and was eluted by imidazole concentration gradient together with the His-tagged UGT1A9, suggesting the formation of stable dimers that contain one His-tagged and one HA-tagged UGT1A9 monomers.
8种人肝脏UDP-葡萄糖醛酸基转移酶（UGTs）在杆状病毒感染的昆虫细胞中表达为融合蛋白，这些融合蛋白带有以6个组氨酸残基（His标签）结尾的短C末端延伸。重组UGT1A1、UGT1A3、UGT1A4、UGT1A6、UGT2B4、UGT2B7和UGT2B15的活性几乎被0.2%的 Triton X-100完全抑制。然而，对于UGT1A9，α-萘酚和东莨菪素的葡萄糖醛酸化对这种抑制具有抗性，而恩他卡朋和其他几种苷元的葡萄糖醛酸化则敏感。带有His标签的UGT1A9通过固定化金属螯合色谱法（IMAC）进行纯化。纯化后的UGT1A9以高速率将东莨菪素葡萄糖醛酸化，而其对恩他卡朋的葡萄糖醛酸化活性较低，且在很大程度上依赖于磷脂的添加。还制备了His标签被血凝素抗原肽（HA标签）取代的重组UGT1A9。昆虫细胞被编码HA标签和His标签的UGT1A9的杆状病毒共同感染。对共同感染细胞的膜，或分别感染细胞的膜混合物进行去污剂提取和IMAC，使用标签特异性抗体分析所得级分中每种类型UGT1A9的存在情况。在单独感染的情况下，带有HA标签的UGT1A9不与柱结合。然而，当与带有His标签的UGT1A9共同感染时，部分带有HA标签的酶与柱结合，并与带有His标签的UGT1A9一起通过咪唑浓度梯度洗脱，这表明形成了稳定的二聚体，其中包含一个带有His标签和一个带有HA标签的UGT1A9单体。
