Collén A, Ward M, Tjerneld F, Stålbrand H
Department of Biochemistry, Lund University, Sweden.
J Chromatogr A. 2001 Mar 2;910(2):275-84. doi: 10.1016/s0021-9673(00)01212-7.
Genetic engineering has been used for fusion of the peptide tag, Trp-Pro-Trp-Pro, on a protein to study the effect on partitioning in aqueous two-phase systems. As target protein for the fusions the cellulase, endoglucanase I (endo-1,4-beta-Dglucan-4-glucanohydrolase, EC 3.2.1.4, EGI, Cel7B) of Trichoderma reesei was used. For the first time a glycosylated two-domain enzyme has been utilized for addition of peptide tags to change partitioning in aqueous two-phase systems. The aim was to find an optimal fusion localization for EGI. The peptide was (1) attached to the C-terminus end of the cellulose binding domain (CBD), (2) inserted in the glycosylated linker region, (3) added after a truncated form of EGI lacking the CBD and a small part of the linker. The different constructs were expressed in the filamentous fungus T. reesei under the gpdA promoter from Aspergillus nidulans. The expression levels were between 60 and 100 mg/l. The partitioning behavior of the fusion proteins was studied in an aqueous two-phase model system composed of the thermoseparating ethylene oxide (EO)-propylene oxide (PO) random copolymer EO-PO (50:50) (EO50PO50) and dextran. The Trp-Pro-Trp-Pro tag was found to direct the fusion protein to the top EO50PO50 phase. The partition coefficient of a fusion protein can be predicted with an empirical correlation based on independent contributions from partitioning of unmodified protein and peptide tag in this model system. The fusion position at the end of the CBD, with the spacer Pro-Gly, was shown to be optimal with respect to partitioning and tag efficiency factor (TEF) was 0.87, where a fully exposed tag would have a TEF of 1.0. Hence, this position can further be utilized for fusion with longer tags. For the other constructs the TEF was only 0.43 and 0.10, for the tag fused to the truncated EGI and in the linker region of the full length EGI, respectively.
基因工程已被用于在蛋白质上融合肽标签Trp-Pro-Trp-Pro，以研究其对在双水相系统中分配行为的影响。作为融合的目标蛋白，使用了里氏木霉的纤维素酶内切葡聚糖酶I（内切-1,4-β-D-葡聚糖-4-葡聚糖水解酶，EC 3.2.1.4，EGI，Cel7B）。首次利用一种糖基化的双结构域酶来添加肽标签，以改变其在双水相系统中的分配行为。目的是为EGI找到最佳的融合定位。该肽（1）连接到纤维素结合结构域（CBD）的C末端，（2）插入糖基化的连接区，（3）添加到缺少CBD和一小部分连接区的截短形式的EGI之后。不同的构建体在构巢曲霉的gpdA启动子控制下在丝状真菌里氏木霉中表达。表达水平在60至100mg/l之间。在由热分离的环氧乙烷（EO）-环氧丙烷（PO）无规共聚物EO-PO（50:50）（EO50PO50）和葡聚糖组成的双水相模型系统中研究了融合蛋白的分配行为。发现Trp-Pro-Trp-Pro标签可将融合蛋白导向顶部的EO50PO50相。在该模型系统中，基于未修饰蛋白和肽标签分配的独立贡献，通过经验相关性可以预测融合蛋白的分配系数。结果表明，在CBD末端带有间隔子Pro-Gly的融合位置在分配方面是最佳的，标签效率因子（TEF）为0.87，而完全暴露的标签的TEF为1.0。因此，该位置可进一步用于与更长的标签融合。对于其他构建体，对于融合到截短的EGI和全长EGI连接区的标签，TEF分别仅为0.43和0.10。
