一种用于高通量筛选的基于荧光的糖基转移酶检测方法。
A fluorescence-based glycosyltransferase assay for high-throughput screening.
作者信息
Ryu Jihye, Eom Min Sik, Ko Wooseok, Han Min Su, Lee Hyun Soo
机构信息
Department of Chemistry, Sogang University, Seoul 121-742, Republic of Korea.
Department of Chemistry, Chung-Ang University, Seoul 156-756, Republic of Korea.
出版信息
Bioorg Med Chem. 2014 Apr 15;22(8):2571-5. doi: 10.1016/j.bmc.2014.02.027. Epub 2014 Mar 2.
Glycosyltransferases catalyze the transfer of a monosaccharide unit from a nucleotide or lipid sugar donor to polysaccharides, lipids, and proteins in a stereospecific manner. Considerable effort has been invested in engineering glycosyltransferases to diversify sugar-containing drugs. An important requirement for glycosyltransferase engineering is the availability of a glycosyltransferase assay system for high-throughput screening of glycosyltransferase mutants. In this study, a general glycosyltransferase assay system was developed based on an ATP sensor. This system showed submicromolar sensitivity and compatibility with both purified enzymes and crude cell extracts. The assay system will be useful for glycosyltransferase engineering based on high-throughput screening, as well as for general glycosyltransferase assays and kinetics.
糖基转移酶以立体特异性方式催化单糖单元从核苷酸或脂质糖供体转移至多糖、脂质和蛋白质。人们已投入大量精力对糖基转移酶进行工程改造，以实现含糖类药物的多样化。糖基转移酶工程改造的一个重要要求是要有一个用于高通量筛选糖基转移酶突变体的糖基转移酶检测系统。在本研究中，基于一种ATP传感器开发了一种通用的糖基转移酶检测系统。该系统显示出亚微摩尔级的灵敏度，并且与纯化酶和粗细胞提取物均兼容。该检测系统将有助于基于高通量筛选的糖基转移酶工程改造，以及通用的糖基转移酶检测和动力学研究。
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