摘要
目的 :本文为进一步研究蛋白 O- Glc NAc糖基化修饰提供重要资源。方法 :为了探索制备纯化具有生物学活性的重组 OGT的有效方法 ,本文用大肠杆菌和 Hi5昆虫细胞表达并纯化具有活性的重组 OGT。携带人 OGTc DNA的 PET32 a质粒在大肠杆菌中表达出带有 1个 S- Tag的重组人 OGT融合蛋白 ,然后用与 S-蛋白质偶联的琼脂糖凝胶颗粒从细菌裂解液中亲和纯化 OGT融合蛋白。结果 :其纯化的 OGT在电泳中显现单一条带并具有生物活性 ,但其活性在洗脱后丧失。在 Hi5昆虫细胞中表达的鼠肝 OGT带有一 His6 tag,经镍离子螯合柱纯化后 ,其比活性达到 0 .88nmol· min- 1 · mg- 1 OGT。结论 :在这两个表达系统中制备重组 OGT各有利弊。
Objectives: These studies provide methods to prepare recombinant OGT that is an important enzyme source for further investigation of protein O-GlcNAcylation.Methods:In order to obtain biologically active recombinant OGT, we have expressed recombinant OGT in two system(E.Coli and insect cells) and purified it to near homogeneity. A PET32a vector containing human OGT cDNA-S tag was expressed in BL21 E.Coli and the recombinant protein was purified with S-protein conjugated agarose beads.Results:The purified OGT fusion protein displayed as a single band in SDS-polyacrylamide gel and had enzymatic activity, but the activity lost after it was eluted from the agarose beads. Recombinant rat liver OGT targetted with His 6 was expressed in Hi5 insect cells and purified by Hitrap Ni +-chelating chromatography. The eluted OGT had a specific enzymatic activity of 0.88 nmol·min -1·mg -1 OGT.
出处
《南通医学院学报》
2004年第1期1-4,共4页
ACTA Academiae Medicinae Nantong
基金
美国国家卫生研究院(NIH)资助
LiFoundation
Inc.(纽约 )资助
