Lin Shu-Chuan, Lin I-Ping, Chou Wei-I, Hsieh Chen-An, Liu Shi-Hwei, Huang Rong-Yuan, Sheu Chia-Chin, Chang Margaret Dah-Tsyr
Institute of Molecular and Cellular Biology, Department of Life Science, National Tsing Hua University, Hsinchu 300, Taiwan, ROC.
Protein Expr Purif. 2009 Jun;65(2):261-6. doi: 10.1016/j.pep.2009.01.008.
The use of protein fusion tag technology simplifies and facilitates purification of recombinant proteins. In this article, we have found that the starch-binding domain derived from Rhizopus oryzae glucoamylase (RoSBD), a member of carbohydrate-binding module family 21 (CBM21) with raw starch-binding activity, is favorable to be applied as an affinity tag for fusion protein engineering and purification in Escherichia coli and Pichia pastoris systems. To determine suitable spatial arrangement of RoSBD as a fusion handle, enhanced green fluorescent protein (eGFP) was fused to either the N- or C-terminus of the SBD, expressed by E. coli, and purified for yield assessment and functional analysis. Binding assays showed that the ligand-binding capacity was fully retained when the RoSBD was engineered at either the N-terminal or the C-terminal end. Similar results have been obtained with the RoSBD-conjugated phytase secreted by P. pastoris. The effective adsorption onto raw starch and low cost of starch make RoSBD practically applicable in terms of development of a new affinity fusion tag for recombinant protein engineering in an economic manner.
蛋白质融合标签技术的应用简化并促进了重组蛋白的纯化。在本文中，我们发现源自米根霉葡糖淀粉酶（RoSBD）的淀粉结合结构域，它是具有生淀粉结合活性的碳水化合物结合模块家族21（CBM21）的成员，有利于作为亲和标签应用于大肠杆菌和毕赤酵母系统中的融合蛋白工程和纯化。为了确定RoSBD作为融合手柄的合适空间排列，将增强型绿色荧光蛋白（eGFP）融合到SBD的N端或C端，由大肠杆菌表达，并进行纯化以评估产量和进行功能分析。结合试验表明，当RoSBD在N端或C端进行工程改造时，其配体结合能力完全保留。毕赤酵母分泌的RoSBD缀合的植酸酶也获得了类似的结果。对生淀粉的有效吸附和淀粉的低成本使得RoSBD在以经济方式开发用于重组蛋白工程的新型亲和融合标签方面具有实际应用价值。
