Zhou Chengdong, Yan Yanping, Fang Jie, Cheng Beijiu, Fan Jun
Key Laboratory of Crop Biology of Anhui Province, Anhui Agricultural University, 130#, Changjiang West Road, Hefei City, Anhui Province 230036, PR, China.
Microb Cell Fact. 2014 Mar 21;13(1):44. doi: 10.1186/1475-2859-13-44.
Recombinant proteins fused with specific cleavage sequences are widely used as substrate for quantitatively analyzing the activity of proteases. Here we propose a new fusion platform for multiple proteases, by using diaminopropionate ammonia-lyase (DAL) as the fusion protein. It was based on the finding that a fused His6-tag could significantly decreases the activities of DAL from E. coli (eDAL) and Salmonella typhimurium (sDAL). Previously, we have shown that His6GST-tagged eDAL could be used to determine the activity of tobacco etch virus protease (TEVp) under different temperatures or in the denaturant at different concentrations. In this report, we will assay different tags and cleavage sequences on DAL for expressing yield in E. coli, stability of the fused proteins and performance of substrate of other common proteases.
We tested seven different protease cleavage sequences (rhinovirus 3C, TEV protease, factor Xa, Ssp DnaB intein, Sce VMA1 intein, thrombin and enterokinase), three different tags (His6, GST, CBD and MBP) and two different DALs (eDAL and sDAL), for their performance as substrate to the seven corresponding proteases. Among them, we found four active DAL-fusion substrates suitable for TEVp, factor Xa, thrombin and DnaB intein. Enterokinase cleaved eDAL at undesired positions and did not process sDAL. Substitution of GST with MBP increase the expression level of the fused eDAL and this fusion protein was suitable as a substrate for analyzing activity of rhinovirus 3C. We demonstrated that SUMO protease Ulp1 with a N-terminal His6-tag or MBP tag displayed different activity using the designed His6SUMO-eDAL as substrate. Finally, owing to the high level of the DAL-fusion protein in E. coli, these protein substrates can also be detected directly from the crude extract.
The results show that our designed DAL-fusion proteins can be used to quantify the activities of both sequence- and conformational-specific proteases, with sufficient substrate specificity.
与特定切割序列融合的重组蛋白被广泛用作定量分析蛋白酶活性的底物。在此，我们提出了一种用于多种蛋白酶的新型融合平台，该平台使用二氨基丙酸氨裂解酶（DAL）作为融合蛋白。这是基于一个发现，即融合的His6标签可显著降低来自大肠杆菌（eDAL）和鼠伤寒沙门氏菌（sDAL）的DAL的活性。此前，我们已表明His6GST标签化的eDAL可用于在不同温度或不同浓度变性剂条件下测定烟草蚀纹病毒蛋白酶（TEVp）的活性。在本报告中，我们将检测DAL上不同的标签和切割序列，以分析其在大肠杆菌中的表达产量、融合蛋白的稳定性以及作为其他常见蛋白酶底物的性能。
我们测试了七种不同的蛋白酶切割序列（鼻病毒3C、TEV蛋白酶、因子Xa、Ssp DnaB内含肽、Sce VMA1内含肽、凝血酶和肠激酶）、三种不同的标签（His6、GST、CBD和MBP）以及两种不同的DAL（eDAL和sDAL）作为七种相应蛋白酶底物的性能。其中，我们发现了四种适用于TEVp、因子Xa、凝血酶和DnaB内含肽的活性DAL融合底物。肠激酶在非预期位置切割eDAL，且不能切割sDAL。用MBP替代GST可提高融合eDAL的表达水平，且该融合蛋白适合作为分析鼻病毒3C活性的底物。我们证明，使用设计的His6SUMO - eDAL作为底物时，带有N端His6标签或MBP标签的SUMO蛋白酶Ulp1表现出不同的活性。最后，由于DAL融合蛋白在大肠杆菌中的高水平表达，这些蛋白质底物也可直接从粗提物中检测到。
结果表明，我们设计的DAL融合蛋白可用于定量序列特异性和构象特异性蛋白酶的活性，具有足够的底物特异性。
