β-1，4-半乳糖基转移酶的纯化及活性测定

【作者】 钟文涛；

【导师】 龚兴国；

【作者基本信息】 浙江大学 ， 生物化学与分子生物学， 2005， 硕士

【摘要】 β-1,4.半乳糖基转移酶(B4GTase)家族是一类在高尔基体上专一性负责合成β→1,4糖苷键,以对蛋白质,特别是膜蛋白进行糖链修饰的蛋白酶。此外,β4GTase还充当细胞膜的识别因子,参与精卵结合、细胞的生长与迁移、内分泌等过程及自体免疫性疾病调控等。本文就β4GTase的基因家族组成、蛋白质结构与功能、体内定位、测定方法及其生物学功能等研究进展进行了简述。 目前,对于大部分的β4GTase表达系统,由于受到各种因素的限制,仍无法获得大量的高效β4GTase蛋白,这给对β4GTase的进一步研究带来了很大困难。Gong等人建立了以E.coli DH5a菌株为宿主菌的小鼠β4GTase融合表达系统。本研究将对上述表达系统进行再优化,并对融合蛋白酶切、纯化,并对获得的重组目的蛋白进行酶活、催化特征等进行系列分析。 本实验首先抽提质粒pGEX-4T-2-β4GTase,而后将其转化进入另一E.coli菌株BL21(DE3)pLysS,因后者含有质粒pLysS,有助于降低宿主细胞原有蛋白的表达水平,从而提高融合蛋白的表达效率。对大量诱导培养的细胞进行裂解,离心,用谷胱甘肽Sepharose 4B亲和柱对上清进行亲和纯化,获得融合蛋白;用凝血酶对纯化后的融合蛋白进行酶切,以Sephadex G-100柱分离酶切后的混合体系,获得重组目的蛋白β4GTase。最终产率为20mg蛋白/1L培养基。然后,利用pH依赖的苯酚红测定法绘制测定体系的光密度.H~+浓度标准曲线,配合重组蛋白催化反应的光密度.时间曲线,计算得知,重组蛋白的酶活为87.4 U,比活为21.85 U/mg。最后,对重组蛋白催化的一系列特征进行分析,确认重组蛋白的最适底物供体、受体分别为UDP-gal及GlcNAc,其最适pH为8.0,最适反应温度为30℃,最适金属辅基为Mn2~+,并发现反应产物UDP及其类似物UTP、α-乳蛋白均为重组蛋白强烈的抑制剂。更多还原

【Abstract】 β-1,4-Galactosyltransferase (β4GTase) forms a β→1,4glycosidase in the Golgi apparatus, manipulating the modification of the glycoproteins, especially those in the cellular membrane. Besides, β4GTase functions as an important recognition molecule in the procession of development and migration of cells, fertilization, internal secretion, and so on. In this review, the relative researches on the molecular structures of the seven members of human β4GTase gene family, and the functional structure, location in vivo, assays on enzyme activity, and biological functions of human β4GTase I were reported brief.Although the β4GTases of several species have been cloned, with the difficulties in the biological systhesis, the recombinant proteins remained a not so high yielding. Gong et al constructed an IPTG-induced expression syetem of mouse β4GTase in a DH5a cell line. With that achievement, we focused on an optimization of the pro-kyraotic expression system, an infinity purification of the target protein, and a pH-sensitive assay on the enzymatic characteristics of β4GTase using phenol red.In the reseach, the new expression system using BL21(DE3)pLysS cell line showed a higher yielding than that with a DH5a system. After an infinity purification of IPTG-fused protein using glutathione Sepharose 4B gel, a digestion with thrombin, and a siege using Sephadex G-100 gel, the target protein β4GTase was obtained with a yielding efficiency of 200mg per 1 litre LB culture. The enzymatic activity of the targer protein was 21.85 U/mg , and the most suitable donar substrate was UDP-gal, and the donar GlcNAc. Other enzymatic characteristics of β4GTase were checked, too. Besides, one of the products UDP, including its analog UTP, and a-lactalbumin were found to be strong inhibitors to the activity of β4GTase.更多还原