A novel quantification method for serine hydrolases in cellular expression system using fluorophosphonate-biotin probe
A novel quantification method for serine hydrolases in cellular expression system using fluorophosphonate-biotin probe
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DOI：
10.1016/j.ejps.2017.12.016
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发表时间：
2018-03-01
影响因子：
4.6
通讯作者：
Imai, Teruko
中科院分区：
文献类型：
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作者：
Abdel-Daim, Amira;Ohura, Kayoko;Imai, Teruko
在本研究中，我们建立了一种定量蛋白质印迹方法，根据重组丝氨酸水解酶的催化机制来测量其表达水平。选择氟磷酸盐 (FP)-生物素作为通用探针来量化其表达水平，因为 FP 部分通过与活性丝氨酸残基的强化学计量结合来不可逆地抑制丝氨酸水解酶。使用 FP-生物素检测的线性度在三种丝氨酸水解酶上进行了评估；人羧酸酯酶 (CES) 1、丁酰胆碱酯酶和猪肝酯酶 (PLE)。从这些酶的等摩尔浓度中获得了类似的响应信号，并且在 0.4-3.4 pmol/泳道范围内观察到良好的线性 (r(2) > 0.99)。采用PLE验证了该方法的准确度和精密度，回收率为97.1-107.2%，相对标准偏差为5.56%。 PLE 因其高稳定性和商业可用性而被选为校准标准。作为所开发方法的应用，我们测量了 HEK293 细胞匀浆 S9 级分中来自人和食蟹猴的四种重组 CES 同工酶的表达水平。人CES1和CES2以及食蟹猴CES1和CES2的表达水平分别为2.51+/-0.1、1.63+/-0.17、0.79+/-0.09和1.37+/-0.13pmol/5μg S9蛋白。根据这些测定，准确评估了它们的水解活性。食蟹猴 CES 对硝基苯酯的水解活性低于人类 CES。两个物种中CES2同工酶的水解酶活性均高于CES1。在所有 CES 同工酶中，观察到丁酸对硝基苯酯的水解速度比乙酸对硝基苯酯快三到五倍，但食蟹猴 CES1 除外，两种底物的水解作用几乎相同。所提供的方法可广泛用于重组丝氨酸水解酶的通用定量分析。
In the present study, we established a quantitative western blotting method to measure the expression level of recombinant serine hydrolases based on their catalytic mechanism. Fluorophosphonate (FP)-biotin was selected as a universal probe to quantify their expression levels, since FP moiety irreversibly inhibits serine hydrolases through strong stoichiometric binding to active serine residue. The linearity of detection using FP-biotin was assessed on three serine hydrolases; human carboxylesterase (CES) 1, butyrylcholinesterase and porcine liver esterases (PLE). Similar response signals were obtained from the equimolar concentrations of these enzymes and excellent linearity was observed at the range of 0.4-3.4 pmol/lane (r(2) > 0.99). Accuracy and precision of the proposed method were proved using PLE with recovery of 97.1-107.2% and relative standard deviation of 5.56%. PLE was selected as a calibration standard because of its high stability and commercial availability. As an application of the developed method, we measured the expression levels of four recombinant CES isozymes from human and cynomolgus macaque in S9 fraction of HEK293 cell homogenates. The expression levels of human CES1 and CES2, and cynomolgus macaque CES1 and CES2 were 2.51 +/- 0.1, 1.63 +/- 0.17, 0.79 +/- 0.09 and 1.37 +/- 0.13 pmol/5 mu g S9 protein, respectively. Based on these determinations, their hydrolytic activities were accurately assessed. Cynomolgus CESs showed lower hydrolysis activities for p-nitrophenyl esters than human CESs. The hydrolase activities of CES2 isozymes were higher than CES1 in both species. Three to five folds faster hydrolysis for p-nitrophenyl butyrate than p-nitrophenyl acetate was observed in all CES isozymes except of cynomolgus CES1 that showed nearly same hydrolysis for both substrates. The provided method could be widely used for universal quantitative analysis of recombinant serine hydrolases.