Chang Kyung Hwa, Baek Nam In, Yang Jai Myung, Lee Jong Min, Bo Jeon Hwang, Chung In Sik
Department of Genetic Engineering, Kyung Hee University, Suwon 449-701, Republic of Korea.
Protein Expr Purif. 2005 Dec;44(2):87-93. doi: 10.1016/j.pep.2005.08.010. Epub 2005 Sep 13.
Beta-Secretase (betaSEC) was expressed in Trichoplusia ni BTI Tn5B1-4 (Tn5B1-4) cells transformed with cDNAs encoding beta1,4-galactosyltransferase (GalT) and Gal beta1,4-GlcNAc alpha 2,6-sialyltransferase (ST). The apparent molecular weight of recombinant beta-secretase was increased from 57 to 59 k Da. A lectin blot analysis indicated that recombinant beta-secretase from Tn5B1-4 betaSEC/GalT-ST cells (Tn5B1-4 cells co-transformed with cDNAs encoding beta-secretase, glycosyltransferases, GalT, and ST) contained the glycan residues of beta1,4-linked galactose and alpha2,6-linked sialic acid. Two-dimensional electrophoresis revealed that recombinant beta-secretase from Tn5B1-4 beta SEC/GalT-ST cells had a lower isoelectric point than beta-secretase from control Tn5B1-4 betaSEC cells (Tn5B1-4 cells transformed only with beta-secretase cDNA). The enzyme activity of recombinant beta-secretase from Tn5B1-4 betaSEC/GalT-ST cells was enhanced up to 77% compared to control Tn5B1-4 betaSEC cells. The concentrations at half-maximum inhibition (IC(50)) values estimated from inhibition analyses using purified beta-secretases from Tn5B1-4/betaSEC and Tn5B1-4/betaSEC/GalT-ST cells were 32 and 290 nM, respectively.
β-分泌酶（βSEC）在转染了编码β1,4-半乳糖基转移酶（GalT）和Galβ1,4-GlcNAcα2,6-唾液酸转移酶（ST）的cDNA的粉纹夜蛾BTI Tn5B1-4（Tn5B1-4）细胞中表达。重组β-分泌酶的表观分子量从57 kDa增加到59 kDa。凝集素印迹分析表明，来自Tn5B1-4 βSEC/GalT-ST细胞（与编码β-分泌酶、糖基转移酶、GalT和ST的cDNA共转染的Tn5B1-4细胞）的重组β-分泌酶含有β1,4-连接的半乳糖和α2,6-连接的唾液酸的聚糖残基。二维电泳显示，来自Tn5B1-4 β SEC/GalT-ST细胞的重组β-分泌酶的等电点低于对照Tn5B1-4 βSEC细胞（仅用β-分泌酶cDNA转染Tn5B1-4细胞）的β-分泌酶。与对照Tn5B1-4 βSEC细胞相比，来自Tn5B1-4 βSEC/GalT-ST细胞的重组β-分泌酶的酶活性提高了77%。使用来自Tn5B1-4/βSEC和Tn5B1-4/βSEC/GalT-ST细胞的纯化β-分泌酶进行抑制分析估计的半数最大抑制浓度（IC50）值分别为32和290 nM。
