高通量蛋白纯化及结晶质量评估的方法学讨论

浙农林—老张

本帖最后由 浙农林—老张 于 2012-11-14 09:05 编辑 High-throughput protein purification and quality assessment for crystallization Midwest Center for Structural Genomics(MCSG)等很多结构基因组学研究所都是这么做的，实现了半自动化高通量蛋白表达纯化和自动化的批量蛋白结晶，具体见方法见附件。Midwest Center for Structural Genomics(MCSG) has developed semi-auto-mated protocols for high-throughput parallel protein expression and purification.A protein,expressed as a fusion with a cleavable affinity tag,is purified in two consecutive immobilized metal affinity chroma-tography(IMAC)steps

                               
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i)the first step is an IMAC coupled with buffer-exchange,or size exclusion chromatography(IMAC-I),followed by the cleavage of the affinity tag using the highly specific Tobacco Etch Virus(TEV)protease[1];the second step is IMAC and buffer exchange(IMAC-II)to remove the cleaved tag and tagged TEV protease.These protocols have been implemented on multidimensional chromatography workstations .

                               
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浙农林—老张

The MCSG has tested the efficacy of two such salvage methods,reduc-tive methylation and in situ proteolysis,on hundreds of different proteins that were recalcitrant to structure determination.Below,we describe these two approaches that show a 7–8%success rate换载体，换宿主仍然不长晶体的补救措施也有描述，但成功率8%左右。方法1：原位的蛋白酶切 如胰凝乳蛋白酶方法2：减少蛋白表面甲基化 如用dimethylamine-borane complex 做蛋白化学相关修饰以上具体实验步骤见附件。