A new recombinant protein expression system for high-throughput screening in the yeast Yarrowia lipolytica
A new recombinant protein expression system for high-throughput screening in the yeast Yarrowia lipolytica
复制标题
DOI：
10.1016/j.mimet.2007.06.008
复制
发表时间：
2007-09-01
影响因子：
2.2
通讯作者：
Marty, Alain
中科院分区：
文献类型：
--
作者：
Bordes, Florence;Fudalej, Franck;Marty, Alain
开发高通量真核筛选程序对于通过定向酶进化获得改良酶的成功是重要的。本方法是为酵母脂溶耶氏菌开发的，它是第二个用于此目的的真核宿主。细胞外脂肪酶Lip2作为表达酶，但该系统将很容易调整为其他酶。我们调整并优化了96孔微孔板中脂质体Y. polytica蛋白表达的方案。通过构建一个包含zeta对接平台的菌株，可以提高酵母的转化效率和表达盒插入率。整个过程的方差系数由36.3%降至18.9%。变异的主要部分（11.7%）来自特定的脂肪酶酶测定，而与转化、生长和表达步骤有关的变异系数仅为7.2%。无活性无性系率由5.8%降至0.2%。转化效率和可变性都与酵母脂解菌的高通量筛选相兼容。(C) 2007 Elsevier B.V.版权所有
Development of a high-throughput eukaryotic screening procedure is important to increase success in obtaining improved enzymes through directed enzyme evolution. This procedure was developed for the yeast Yarrowia lipolytica which becomes the second eukaryotic host for this purpose. The extracellular lipase Lip2 was used as expressed enzyme but this system will be easily adjusted for other enzymes. We adapted and optimized the protocol for protein expression by Y. lipolytica in 96-well microplates. Yeast transformation efficiency and expression cassette insertion were increased by constructing a strain containing a zeta docking platform for targeted integration into the genome. The coefficient of variance of the full process was reduced from 36.3% to 18.9%. The main part of the variability (11.7%) arises from the specific lipase enzyme assay whereas the coefficient of variance concerning transformation, growth and expression steps represents only 7.2%. The rate of clone with no activity was reduced from 5.8% to 0.2%. Both transformation efficiency and variability are then compatible with high-throughput screening in the yeast Y. lipolytica. (C) 2007 Elsevier B.V. All rights reserved.