抗菌肽Cecropin A的可溶表达及快速纯化工艺研究  

Soluble expression and rapid purification process of the antimicrobial peptide Cecropin A

作　　者：姚欣 邹沛璇 郑永祥 余蓉[1] YAO Xin;ZOU Peixuan;ZHENG Yongxiang;YU Rong(West China School of Pharmacy,Sichuan University,Chengdu,Sichuan,610041 P.R.China)

机构地区：[1]四川大学华西药学院,四川成都610041

出　　处：《华西药学杂志》2025年第2期131-134,共4页West China Journal of Pharmaceutical Sciences

基　　金：国家自然科学基金资助项目(批准号:81973226)。

摘　　要：目的通过促溶标签和His tag实现抗菌肽Cecropin A在原核系统中的可溶表达和分离纯化,并评价其对铜绿假单胞菌的抗菌活性。方法将小分子泛素样修饰蛋白标签SUMO以及His tag与Cecropin A融合,构建pET30a(+)-His-SUMO-cecropin A表达载体,通过一步镍亲和层析捕获His-SUMO-cecropin A,经Ulp1酶切后再通过镍亲和层析流穿得到无氨基酸残留的Cecropin A,采用细菌生长浊度法评价Cecropin A对铜绿假单胞菌的活性。结果在BL21(DE3)和Transetta(DE3)感受态中均能表达His-SUMO-cecropin A蛋白,与SUMO标签融合成促进了Cecropin A在上清中的可溶表达。经两步镍亲和层析,可纯化得到0.447 mg·mL^(-1)的Cecropin A,3μmol·L^(-1)的Cecropin A与铜绿假单胞菌作用1 h后的ΔOD_(600)%为66.80%,Cecropin A对铜绿假单胞菌有抗菌活性。结论通过与His-SUMO标签融合,可提高抗菌肽Cecropin A的可溶性,有利于蛋白纯化,纯化的Cecropin A对铜绿假单胞菌有较好的抗菌活性。OBJECTIVE To express antibacterial peptide Cecropin A,to purity it in a prokaryotic system using a solubilizing tag and His tag,and its antibacterial efficacy against Pseudomonas aeruginosa.METHODS The pET30a(+)-His-SUMO-cecropin A expression vector was engineered by fusing the small ubiquitin-like modifier(SUMO)and His tag to Cecropin A,allowing efficient capture of His-SUMO-cecropin A through one-step nickel affinity chromatography.After Ulp1 enzyme digestion,Cecropin A without any amino acid residues was obtained through flow-through nickel affinity chromatography.The antibacterial activity of Cecropin A against P.aeruginosa was assessed using the bacterial growth turbidity assay.RESULTS His-SUMO-cecropin A protein could be expressed in both BL21(DE3)and Transetta(DE3)competent cells,and fusion with the SUMO promotd the soluble expression of Cecropin A in supernatant.Cecropin A at 0.447 mg·mL^(-1)was purified by two-step nickel affinity chromatography.TheΔOD600%of cecropin A at 3μmol·L^(-1)was 66.80%after 1 h interaction with P.aeruginosa.Cecropin A demonstrated antibacterial activity against P.aeruginosa.CONCLUSION Via fusion with His-SUMO,the solubility of Cecropin A can be improved,which is conducive to protein purification,and the purified Cecropin A has good antibacterial activity against P.aeruginosa.

关 键 词：天蚕素抗菌肽 Cecropin A 可溶表达 促溶标签 SUMO His tag 融合蛋白 铜绿假单胞菌 

分 类 号：R915[医药卫生—微生物与生化药学]