基因工程表达内含肽介导的抗菌肽(MME)及其酰胺化

Intein-mediated Expression in E.coli and Amidation of Antimicrobial peptide MME

【目的】探讨在巯乙基磺酸钠(MESNa)与碳酸氢铵(NH4HCO3)存在下,对在大肠杆菌上表达的重组融合蛋白中内含肽(Intein)用硫醇(DTT)进行自剪切,促使抗菌肽(MME)碳末端实现酰胺化。【方法】构建用大肠杆菌表达内含肽介导的MME重组质粒,通过表达获得依次由"组氨酸、Sumo标签、MME、内含肽"构成的融合蛋白,用镍柱及透析进行纯化,在MESNa和NH4HCO3存在下,用DTT对内含肽进行自剪切,促使MME碳末端酰胺化,肠激酶切割、纯化,得到碳末端酰胺化的MME。质谱和二级串联质谱配合使用,检测MME及其碳末端酰胺化。【结果】通过PCR,鉴定融合蛋白,其基因片段为837 bp,符合预期。质谱鉴定得MME相对分子量为3057.7与其理论值3057.64一致。二级串联质谱检测得MME碳端碎片的分子量为1214.728,与碳末端酰胺化的MME碳端碎片理论分子量1214.739相符,匹配度为45,表明本研究制备的MME碳末端已被酰胺化,该蛋白为可溶。【结论】用大肠杆菌成功地表达了重组融合蛋白,在MESNa与NH4HCO3存在下,促进了内含肽自剪切,MME碳末端被酰胺化,实现了简单的"一步法"制备。质谱与二级串联质谱配合使用,可灵敏、简单地对碳末端酰胺化多肽进行检测,可作为该项检测方法的一种选择,结果表明,本研究成功地制备了碳末端酰胺化的MME。

【Objective】Efficient method to amidate the carbon-terminal of the antimicrobial peptide MME through selfsplitting intein in recombination fusion proteins expressed in E.coli with DTT in the presences of MESNa and NH4HCO3 was explored.【Method】Recombinant plasmid of MME was constructed to induce the intein-mediated expression in E.coli to successively obtain fusion proteins composed of histidine,sumo label,target polypeptide,and intein.The proteins were subsequently purified in a process using a nickel column and dialysis.With MESNa and NH4HCO3,an intein self-split procedure was completed by DTT to amidate the carbon-terminal on MME,then cut and purify the intestinal kinase.Both MME and the amidation were verified by the standard mass spectrometry and two-stage,tandem mass spectrometry.【Result】The resultant gene fragment of the fusion protein was determined by PCR to be 837 bp long as expected.In comparison to the theoretical value of 3057.64,the relative molecular weight of MME obtained by the standard mass spectrometry was 3057.7.The molecular weight of the amidated carbon-terminal fragments of MME was measured by the two-stage,tandem mass spectrometry to be 1,214.728,which was close to the known theoretical value of 1214.739 with a matching rate of 45.It appeared that the carbon-terminal of MME had been effectively amidated,and the protein obtained was soluble.【Conclusion】The recombinant fusion proteins were successfully expressed in E.coli to enable the intein selfsplitting in the presences of MESNa and NH4HCO3 and amidate the carbon terminal of MME.Therefore,the current simple,one-step preparation could achieve accurate,duplicatable,and meaningful results.By combining the standard mass spectrometry and two-stage,tandem mass spectrometry,the amidated carbon-terminal polypeptides could satisfactorily be detected.