¦¢-1, 4 - Galactosyltransferase Enzyme Purification And Activity Determination

β-1,4-Galactosyltransferase (β4GTase) forms a βâ†'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.