Vernet T, Chatellier J, Tessier D C, Thomas D Y
Biotechnology Research Institute, National Research Council of Canada, Montréal, Québec.
Protein Eng. 1993 Feb;6(2):213-9. doi: 10.1093/protein/6.2.213.
A microbial expression system for the study of the cysteine protease papain has been developed as a more useful alternative to the insect cell/baculovirus expression system we have previously used. A synthetic papain precursor (propapain) gene was expressed in the yeast Saccharomyces cerevisiae under the control of the alpha-factor promoter. Efficient expression required fusion of the propapain sequence with the yeast alpha-factor prepro region and a yeast host cell defective in the synthesis of vacuolar proteases. Surprisingly, the glycosylated form of the inactive papain precursor is not secreted, but accumulates within the yeast cell. Complete conversion of the intracellular zymogen into active mature papain could be achieved in vitro. Purified recombinant papain produced by the yeast system has kinetic characteristics similar to those of the natural enzyme. An advantage of the yeast expression system over the baculovirus/insect cell system is that we can perform mutagenesis and screening of papain mutants very efficiently. We have set up a 'one-tube' screening procedure for the simultaneous characterization of numerous mutants of the papain precursor. Yeast cells are grown and lysed in microtiter plate wells and the released papain precursor is then activated to mature papain. This assay allows easy discrimination between proteins with close to wild type properties and proteins that are not functional. We have applied this assay to investigate the spectrum of amino acids which are tolerated at Asn175 of papain using two independently derived libraries of mutants at this position. Many amino acid substitutions at this position are not accepted; only the reintroduction of Asn restored normal function.
已开发出一种用于研究半胱氨酸蛋白酶木瓜蛋白酶的微生物表达系统，作为我们之前使用的昆虫细胞/杆状病毒表达系统更有用的替代方案。合成的木瓜蛋白酶前体（酶原木瓜蛋白酶）基因在α-因子启动子的控制下在酿酒酵母中表达。高效表达需要将酶原木瓜蛋白酶序列与酵母α-因子前原区融合，并且酵母宿主细胞在液泡蛋白酶的合成方面存在缺陷。令人惊讶的是，无活性木瓜蛋白酶前体的糖基化形式不会分泌，而是在酵母细胞内积累。细胞内酶原完全转化为活性成熟木瓜蛋白酶可在体外实现。酵母系统产生的纯化重组木瓜蛋白酶具有与天然酶相似的动力学特性。酵母表达系统相对于杆状病毒/昆虫细胞系统的一个优点是，我们可以非常有效地进行木瓜蛋白酶突变体的诱变和筛选。我们已经建立了一种“单管”筛选程序，用于同时表征木瓜蛋白酶前体的众多突变体。酵母细胞在微量滴定板孔中生长并裂解，然后将释放的木瓜蛋白酶前体激活为成熟木瓜蛋白酶。该测定法允许轻松区分具有接近野生型特性的蛋白质和无功能的蛋白质。我们已应用该测定法，使用在该位置独立衍生的两个突变体文库，研究木瓜蛋白酶Asn175处可耐受的氨基酸谱。该位置的许多氨基酸取代不被接受；只有重新引入Asn才能恢复正常功能。
