重组LysargiNase蛋白酶的表达纯化及其在蛋白质组学层面的分析

Expression and Purification of Recombinant LysargiNase and Its Application in Proteomic Analysis

在蛋白质组学中,胰蛋白酶具有最优秀的酶切特异性及最广泛的应用,然而胰蛋白酶在特定研究中作用仍然有限。作为胰蛋白酶镜像酶,LysargiNase能够特异性切割蛋白质或多肽中赖氨酸与精氨酸位点的氨基端肽键,被认为具有部分替代胰蛋白酶的潜力。为系统分析LysargiNase在蛋白质组学中的应用价值,本研究设计出带有His-tag标签的重组质粒,并在原核表达系统中顺利表达出重组LysargiNase蛋白酶,经测试具有预期活性及酶切特异性。将重组LysargiNase与胰蛋白酶的酶切结果进行对比,显示两种蛋白酶的酶切结果存在很高的互补性,LysargiNase酶能够提高胰蛋白酶酶切的鉴定序列覆盖度。对比重组LysargiNase与两种商品化产品的酶切效率,结果显示3种蛋白酶的性能相近,乙酰化样品的酶切位点识别特异性极高。此外,综合评价几种化学修饰对LysargiNase酶切效率的影响,发现酶原水平的双甲基化修饰与活化水平的乙酰化修饰均能够提高LysargiNase的酶切效率。

In proteomic studies,trypsin has the best digestion specificity and the most widespread application,but it is still limited in specific research.LysargiNase,a protease mirrors trypsin in specificity,cleaves N-terminal peptide bonds of lysine and arginine in proteins or peptides,and has potential to partially replace trypsin.To systematically analyze the proteomic application value of LysargiNase,a His-tagged recombinant plasmid was designed in this study,and recombinant LysargiNase was successfully expressed in prokaryotic expression system,which had the expected activity and digestion specificity.Comparing the digestion results of recombinant LysargiNase with trypsin,it was shown that the digestion results of two proteases were highly complementary,and LysargiNase could improve the sequence coverage identified by trypsin digestion.Comparing the digestion efficiency of recombinant LysargiNase with two commercial products,the results showed that these three proteases were comparable,and the site recognition specificity when digesting acetylated samples were particularly high.In addition,when systematically evaluating the effects of several chemical derivatization on the digestion efficiency of LysargiNase,it was revealed that dimethylation of pro-LysargiNase and acetylation of activated LysargiNase could enhance the digestion efficiency.