Vernet T, Tessier D C, Richardson C, Laliberté F, Khouri H E, Bell A W, Storer A C, Thomas D Y
Genetic Engineering Section, National Research Council of Canada, Montreal, Quebec.
J Biol Chem. 1990 Sep 25;265(27):16661-6.
The synthetic gene coding for the precursor of the cysteine protease papain (EC 3.4.22.2) has been expressed using the baculovirus/insect cell system. The prepropapain gene was cloned into the transfer vector IpDC125 behind the polyhedrin promoter. The recombinant construct was then incorporated by homologous recombination into the Autographa californiaca nuclear polyhedrosis virus genome. The host Spodoptera frugiperda Sf9 cells infected with the recombinant baculovirus secrete an enzymatically inactive N-glycosylated papain precursor. This zymogen could be activated in vitro to yield about 400 nmol of active papain per liter of culture. The recombinant active mature papain was enzymatically indistinguishable from natural papain but the precursor was not processed to the same amino acid residue. The insect cells also accumulated prepropapain and glycosylated propapain intracellularly. This accumulation was an indication that there are rate-limiting steps in the secretion of proteins from insect cells in this expression system. Characterization of mutants of the precursor has shown that entry into the secretory pathway and addition of carbohydrate are prerequisite conditions for the production and secretion of functional propapain.
编码半胱氨酸蛋白酶木瓜蛋白酶（EC 3.4.22.2）前体的合成基因已通过杆状病毒/昆虫细胞系统进行表达。前木瓜蛋白酶基因被克隆到多角体蛋白启动子下游的转移载体IpDC125中。然后通过同源重组将重组构建体整合到苜蓿银纹夜蛾核型多角体病毒基因组中。用重组杆状病毒感染的宿主草地贪夜蛾Sf9细胞分泌一种无酶活性的N-糖基化木瓜蛋白酶前体。这种酶原可在体外被激活，每升培养物可产生约400 nmol的活性木瓜蛋白酶。重组活性成熟木瓜蛋白酶在酶学性质上与天然木瓜蛋白酶无法区分，但前体的加工氨基酸残基不同。昆虫细胞还在细胞内积累了前木瓜蛋白酶和糖基化的木瓜蛋白酶原。这种积累表明在该表达系统中昆虫细胞分泌蛋白质存在限速步骤。对前体突变体的表征表明，进入分泌途径和添加碳水化合物是产生和分泌功能性木瓜蛋白酶原的先决条件。
