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首页> 外文期刊>Acta crystallographica, Section F. Structural biology and crystallization communications >Heterologous expression, purification, crystallization and preliminary X-ray analysis of Trichoderma reesei xylanase II and four variants
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Heterologous expression, purification, crystallization and preliminary X-ray analysis of Trichoderma reesei xylanase II and four variants

机译：里氏木霉木聚糖酶II及其四个变体的异源表达，纯化，结晶和初步X射线分析

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摘要

Xylanase II from Trichoderma reesei catalyzes the hydrolysis of glycosidic bonds in xylan. Crystallographic studies of this commercially important enzyme have been initiated to investigate its reaction mechanism, substrate binding and dependence on basic pH conditions. The wild-type protein was heterologously expressed in an Escherichia coli host using the defined medium and four active-site amino acids were replaced to abolish its activity (E177Q and E86Q) or to change its pH optimum (N44D and N44H). Cation-exchange and size-exclusion chromatography were used to obtain >90% protein purity. The ligand-free proteins and variant complexes containing substrate (xylohexaose) or product (xylotriose) were crystallized in several different space groups and diffracted to high resolutions (from 1.07 to 1.55 angstrom).
机译：来自里氏木霉（Trichoderma reesei）的木聚糖酶II催化木聚糖中糖苷键的水解。已经开始对该商业上重要的酶进行晶体学研究，以研究其反应机理，底物结合和对碱性pH条件的依赖性。使用定义的培养基在大肠杆菌宿主中异源表达野生型蛋白，并替换了四个活性位点氨基酸以取消其活性（E177Q和E86Q）或更改其最佳pH（N44D和N44H）。阳离子交换色谱法和尺寸排阻色谱法用于获得> 90％的蛋白纯度。不含底物（木己糖）或产物（木三糖）的不含配体的蛋白质和变体复合物在几个不同的空间组中结晶，并衍射至高分辨率（1.07至1.55埃）。

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