Recombinant expression, purification and characterization of acetylated LysargiNase from Escherichia coli with high activity and stability
Recombinant expression, purification and characterization of acetylated LysargiNase from Escherichia coli with high activity and stability
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具有高活性和稳定性的大肠杆菌乙酰化 LysargiNase 的重组表达、纯化和表征
DOI：
10.1002/rcm.8440
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发表时间：
2019
影响因子：
2
通讯作者：
Xu Ping
中科院分区：
文献类型：
--
作者：
Zhang Junling;Zhao Mingzhi;Xiao Weidi;Chang Lei;Wang Fuqiang;Xu Ping
RationaleLysargiNase是一种新型的金属蛋白酶，能切割赖氨酸或精氨酸残基的N末端。LysargiNase产生的多肽只是胰酶产生的多肽的镜像。LysargiNase的这些特性为基于MS的蛋白质组学研究提供了强有力的工具。用一种简单、廉价的方法生产出活性高、稳定性好的溶酶，将极大地促进蛋白质组学的研究。方法将N端带有肠激酶酶切位点的LysargiNase编码序列插入表达载体pGEX-4 T-2，转化E.ColiBL21(DE3)。在工作体积为5 L的14-L发酵罐中培养，加入终浓度为1 mM的异丙基-D-硫代半乳糖苷(IPTG)诱导蛋白表达。将重组LysargiNase负载到GSTrap上，柱上消化去除GST标签，然后通过层析纯化得到纯化的重组LysargiNase。用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法和LC/MS/MS法比较了重组溶血酶和乙酰化溶血酶与简单蛋白质底物人血清白蛋白和复杂蛋白质组样品酵母裂解物的酶切效率和特异性。结果重组溶血酶在pGEX-4 T-2中得到了高效表达。ColiBL21(DE3)。此外，纯化的LysargiNase的乙酰化显著提高了其抗自溶能力，从而使蛋白质组学样品得到更完全的消化，并获得更多的LC/MS/MS鉴定的多肽和蛋白质。结论本研究构建了一种高效表达系统，用于生产重组LysargiNase。这在下游的纯化过程中具有很大的优势。我们还证实了乙酰化修饰可以提高重组LysargiNase的稳定性和活性。这项研究为蛋白质组学研究提供了一个更好的方法来生产这个强大的工具。
RationaleLysargiNase is a novel characterized metalloprotease that can cleave the N‐terminii of lysine or arginine residues. The peptides generated by LysargiNase are just mirrors to those generated by trypsin. These characteristics of LysargiNase provide a powerful tool for mass spectrometry (MS)‐based proteomics research. A highly active and stable LysargiNase produced by an easy and inexpensive method could greatly benefit proteomics research. Here, we report the soluble recombinant expression, purification and acetyl modification of LysargiNase inEscherichia coli.MethodsThe coding sequence of LysargiNase with an enterokinase cleavage site at the N‐terminus was inserted into plasmid pGEX‐4 T‐2 and transformed intoE. coliBL21 (DE3). The strain was cultured in a 14‐L fermenter with a working volume of 5 L. The protein expression was induced by adding isopropyl‐β‐D‐thiogalactoside (IPTG) to a final concentration of 1 mM. The recombinant LysargiNase was loaded onto a GSTrap and an on‐column digestion was performed to remove the GST tag and was subsequently purified by chromatographic purification.In vitroacetylation of LysargiNase was performed by using acetic anhydride. The digestion efficiency and specificity of recombinant LysargiNase and acetylated LysargiNase were compared with simple protein substrate, human serum albumin (HSA), and a complex proteomic sample, yeast lysate, by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) and liquid chromatography/tandem mass spectrometry (LC/MS/MS).ResultsHighly soluble expression of recombinant LysargiNase was achieved by plasmid pGEX‐4 T‐2 inE. coliBL21 (DE3). In addition, acetylation of purified LysargiNase significantly increased its resistance to autolysis, which resulted in a more complete digestion of proteomics samples and more identified peptides and proteins by LC/MS/MS.ConclusionsIn this study, we constructed a highly soluble expression system for producing recombinant LysargiNase inE. coli, which gave tremendous advantages in the downstream purification process. We also confirmed that acetyl modification can increase the stability and activity of recombinant LysargiNase. The study provided a superior way to produce this powerful tool for proteomics research.