Collén A, Ward M, Tjerneld F, Stålbrand H
Department of Biochemistry, Lund University, PO Box 124, S-221 00 Lund, Sweden.
J Biotechnol. 2001 May 4;87(2):179-91. doi: 10.1016/s0168-1656(01)00241-3.
Endoglucanases (endo-1,4-beta-D-glucan-4-glucanohydrolase, EC 3.2.1.4) are industrially important enzymes. In this study endoglucanase I (EGI or Cel7B) of the filamentous fungi Trichoderma reesei has been genetically engineered to investigate the influence of tryptophan rich peptide extensions (tags) on partitioning in an aqueous two-phase model system. EGI is a two-domain enzyme and is composed of a N-terminal catalytic domain and a C-terminal cellulose binding domain, separated by a linker. The aim was to find an optimal tag and fusion position, which further could be utilised for large scale extractions. Peptide tags of different length and composition were attached at various localisations of EGI. The fusion proteins were expressed from T. reesei with the use of the gpdA promoter from Aspergillus nidulans. Variations in secreted levels between the engineered proteins were obtained. The partitioning of EGI in an aqueous two-phase system composed of a thermoseparating ethylene oxide-propylene oxide random copolymer (EO(50)PO(50)) and dextran, could be significantly improved by relatively minor genetic engineering. The (Trp-Pro)(4) tag added after a short stretch of the linker, containing five proline residues, gave in the highest partition coefficient of 12.8. The yield in the top phase was 94%. The specific activity was 83% of the specific activity of unmodified EGI on soluble substrate. The efficiency of a tag fused to a protein is shown by the tag efficiency factor (TEF). A hypothetical TEF of 1.0 would indicate full tag exposure and optimal contribution to the protein partitioning by the fused tag. The location of the fusion point after the sequence of five proline residues in the linker of EGI is the most beneficial in two-phase separation. The highest TEF (0.97) was obtained with the (Trp-Pro)(2) tag at this position, indicating full exposure and intactness of the tag. However, the peptide tag composed of (Trp-Pro)(4) improved the partition properties the most but had lower TEF in comparison to (Trp-Pro)(2).
内切葡聚糖酶（内切-1,4-β-D-葡聚糖-4-葡聚糖水解酶，EC 3.2.1.4）是具有重要工业价值的酶。在本研究中，对丝状真菌里氏木霉的内切葡聚糖酶I（EGI或Cel7B）进行了基因工程改造，以研究富含色氨酸的肽段延伸（标签）对其在水相双相模型系统中分配的影响。EGI是一种双结构域酶，由一个N端催化结构域和一个C端纤维素结合结构域组成，二者由一个连接子隔开。目的是找到一个最佳标签和融合位置，以便进一步用于大规模提取。将不同长度和组成的肽标签连接到EGI的不同位置。利用构巢曲霉的gpdA启动子从里氏木霉中表达融合蛋白。获得了工程蛋白分泌水平的差异。通过相对较小的基因工程改造，可显著改善EGI在由热分离环氧乙烷-环氧丙烷无规共聚物（EO(50)PO(50)）和葡聚糖组成的水相双相系统中的分配。在连接子的一段短序列（含有五个脯氨酸残基）之后添加的(Trp-Pro)(4)标签，给出了最高分配系数12.8。上相中的产率为94%。对可溶性底物的比活性是未修饰EGI比活性的83%。标签效率因子（TEF）显示了融合到蛋白质上的标签的效率。假设的TEF为1.0表明标签完全暴露且对融合标签的蛋白质分配有最佳贡献。在EGI连接子中五个脯氨酸残基序列之后的融合点位置在双相分离中最有利。在此位置使用(Trp-Pro)(2)标签获得了最高TEF（0.97），表明标签完全暴露且完整。然而，由(Trp-Pro)(4)组成的肽标签对分配特性的改善最大，但与(Trp-Pro)(2)相比TEF较低。
