抗菌肽-裂解酶融合抗铜绿假单胞菌蛋白的可溶表达与活性评价
Soluble expression and activity evaluation of antibacterial peptide-endolysin fusion anti-pseudomonas aeruginosa protein
周俊秀 1姚欣 1郑永祥 1余蓉1
作者信息
- 1. 四川大学华西药学院,四川成都 610041
- 折叠
摘要
目的 通过与促溶蛋白融合表达,分离、纯化获得抗菌融合蛋白,并初步评价其抗菌活性.方法 将绵羊骨髓细胞抗菌肽SMAP29的N端与源自噬菌体JD010裂解酶LysPA26的C端融合设计新的抗菌蛋白(AL).通过同源重组构建pCold-His-TF-AL表达质粒.通过Ni-亲和层析和阴离子交换层析分离纯化AL,采用平板计数法初步评价AL的抗铜绿假单胞菌活性.结果 在E.coli BL21(DE3)中,质粒pCold-His-TF-AL能明显表达出抗菌蛋白AL,并且90％以上的AL为可溶形式分布于E.coli破菌液的上清液中.可分离纯化得到纯度超95％的AL.0.05 mg·mL-1 AL在30 min内可将铜绿假单胞菌PAO1菌量降低5.55个对数单位,具较高的抗菌活性.结论 通过采用Trigger Factor标签融合表达和冷休克诱导方法可实现AL的高效可溶表达,纯化的AL对铜绿假单胞菌具有明显的抗菌活性.
Abstract
OBJECTIVE To express and purify an antibacterial peptide-endolysin fusion protein by fusion expression with Trigger Factor,and initially evaluated its antibacterial activity.METHODS A new antibacterial protein AL was designed by fusing 29 amino acids at the N-terminus of the sheep myeloid antibacterial peptide with the C-terminus of endolysin LysPA26.pCold-His-TF-AL expression plasmid was constructed by homologous recombination.Antibacterial protein AL was purified by Ni-affinity chromatography and anion-exchange chromatography,and the anti-Pseudomonas aeruginosa activity of AL was initially evaluated by plate counting method.RESULTS In E.coli BL21(DE3),the plasmid pCold-His-TF-AL could significantly express the antibacterial protein AL,and more than 90％of AL was distributed in the supernatant of E.coli lysate in the soluble form.The anti-Pseudomonas aeruginosa activity of AL was evaluated by plate counting method,which showed that 0.05 mg·mL-1 AL could reduce the amount of P.aeruginosa PAO1 by 5.55 log units within 30 min,with high antibacterial activity.CONCLUSION Efficient soluble expression of AL can be achieved by using Tigger Factor fusion expression and cold shock induction method,and the purified AL had significant antibacterial activity against P.aeruginosa.
关键词
绵羊骨髓细胞抗菌肽/SMAP29/裂解酶/LysPA26/重组蛋白/TF标签/可溶表达/铜绿假单胞菌Key words
Sheep myeloid antibacterial peptide/SMAP29/Lyase/LysPA26/Recombinant protein/Trigger Factor/Soluble expression/Pseudomonas aeruginosa引用本文复制引用
基金项目
国家自然科学基金资助项目(81973226)
四川省自然科学基金面上项目(2023NSFSC0559)
出版年
2024
NETL
NSTL
万方数据