Brömme D, Bonneau P R, Lachance P, Wiederanders B, Kirschke H, Peters C, Thomas D Y, Storer A C, Vernet T
Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec.
J Biol Chem. 1993 Mar 5;268(7):4832-8.
A cDNA encoding the human lysosomal cysteine proteinase cathepsin S precursor has been expressed in yeast using the pVT100-U expression vector containing the alpha-factor promoter. The procathepsin S gene was expressed either as a fusion protein with the pre-region or with the prepro-region of the yeast alpha-factor precursor gene. Following in vitro processing both constructs gave an identical active mature enzyme with a molecular weight of 24,000. After prolonged cultivation of the cells the recombinant protein is also found as an active proteinase in the culture supernatant. The precursor can be activated in vitro at pH 4.5 and 40 degrees C under reducing conditions. The in vitro activated enzyme has a 6-amino acid NH2-terminal extension when compared with the native bovine enzyme. The purified enzyme displays a bell-shaped pH activity profile with a pH optimum of 6.5 and pK values of 4.5 and 7.8. The isoelectric point of the recombinant human cathepsin S is between 8.3 and 8.6 and about 1.5 pH units higher than for the bovine enzyme. The kinetic data for several synthetic substrates and inhibitors reveal a preference for smaller amino acid residues in the binding subsites S2 and S3 of cathepsin S. Like the bovine enzyme, the recombinant human cathepsin S is characterized by a broader range of pH stability (pH 5-7.5) than cathepsins B and L.
利用含有α-因子启动子的pVT100-U表达载体，已在酵母中表达了编码人溶酶体半胱氨酸蛋白酶组织蛋白酶S前体的cDNA。组织蛋白酶S原基因既可以与酵母α-因子前体基因的前导区或前原区作为融合蛋白进行表达。经过体外加工，两种构建体都产生了分子量为24,000的相同活性成熟酶。细胞长时间培养后，重组蛋白也以活性蛋白酶的形式存在于培养上清液中。前体在还原条件下于pH 4.5和40℃可在体外被激活。与天然牛酶相比，体外激活的酶具有6个氨基酸的NH2末端延伸。纯化的酶呈现钟形pH活性曲线，最适pH为6.5，pK值为4.5和7.8。重组人组织蛋白酶S的等电点在8.3至8.6之间，比牛酶高约1.5个pH单位。几种合成底物和抑制剂的动力学数据表明，组织蛋白酶S在结合亚位点S2和S3中更倾向于较小的氨基酸残基。与牛酶一样，重组人组织蛋白酶S的特点是pH稳定性范围（pH 5-7.5）比组织蛋白酶B和L更广。
