(S)-β-没药烯合酶N端截短对其功能的影响

Effect of N-terminal truncation of (S)-β-bisabolene synthase on its function

【目的】探究(S)-β-没药烯合酶N端截短对其功能的影响。【方法】在截短处设计引物,引物引入限制性内切酶酶切位点,PCR产物经双酶切,连接到相应载体,经大肠杆菌Rosetta菌株表达,亲和层析纯化蛋白后蛋白酶切去融合标签,高分辨质谱测定蛋白分子量。体外催化产物经GC-FID/MS分析产物变化,Malachite Green无机磷酸含量法测定表观Kcat。【结果】截短10、20、30、40个氨基酸融合蛋白表达及纯化成功,GCFID分析结果表明去标签的不同长度截短的酶催化主产物均为没药烯,显示活性没有丧失;随着N端截短氨基酸数目的增多,表观Kcat稍有变化。截短60个氨基酸的融合蛋白His-N60表达成功,但亲和层析失败,取表达的上清蛋白体外催化,结果显示无倍半萜合酶活性。【结论】N端截短40个氨基酸不影响其产物特异性和活性。此结果和其他报道不一致,显示不同的倍半萜合酶N端的作用可能不同。

【Objective】This study aims to explore the effect of(S)-β-bisabolene synthase N-terminal truncation on its function.【Methods】According the length of truncation,several 5’end primers with BamHI restriction site and the 3’end primer at the stop condon site were designed to get different length of truncated DNA products by PCR.PCR products were digested with BamHI and XhoI and then ligated into pET15 bvector.Recombinant protein were isolated from E.coli Rosetta(DE3)cells with recombinant plamids and purified by Ni2 +affinity chromatography.After the fused tag digested with HRV3 Cprotease,the exact moleculer mass of truncated enzymes were verified by high-resolution mass spectrometry.The product fromin ivitro catalytic reaction was analyzed by GC-FID/MS,and the apparent Kcat was determined by Malachite Green inorganic phosphoric acid content method.【Results】The N-terminal truncated enzymes lacking 10,20,30,40 residules were all purified successfull.The GC-FID analysis showed that the main product of these enzymes were(S)-β-bisabolene and 10,20,30,40 truncations have no effect on the enzyme activity.The apparent Kcat of theses four enzymes changed sligthely.The 60 residules truncated protein was successfully expressed,but could be not purified by affinity chromatography.The crude protein was used to in ivitro catalytic reaction and the result showed that no sesquiterpene synthase activity was found.【Conclusion】The 40 residules N-terminal truncation of(S)-β-bisabolene synthase does not affect its product specificity and activity.This result is inconsistent with other reports,suggesting that the function of different sesquiterpene synthases on the N-terminus may be different.