表面活性肽在大肠杆菌中的异源表达

为构建新型表面活性肽的表达载体，并探讨其在大肠杆菌中的表达及表达产物的纯化，将编码新型活性肽A₆K的重复DNA片段(A₆K)₁₅分别插入到4中不同的原核表达载体中，经酶切和测序验证后，转化到3种不同的表达宿主进行表达，通过改变异丙基-β-D-硫代半乳糖苷(isopropyl-β-D-thiogalactoside, IPTG)浓度、诱导温度和时间来优化表达条件。表达产物通过镍-次氮基三乙酸(nickel-nitrilotriacetic acid, Ni-NTA)螯合层析进行纯化，通过(A₆K)₁₅上的6×His与抗体特异性结合进行Western blot分析，用二喹啉甲酸(bicinchoninic acid, BCA)法测定产物浓度，进行经济效益分析。构建了含有目的基因(A₆K)₁₅的重组表达质粒，筛选出最优载体pET-28a-SUMO-(A₆K)₁₅和最优宿主BL21(DE3)。当IP...

Heterologous expression of surfactant-like peptides in Escherichia coli

In this study, we investigated the expression of a novel surfactant-like peptide(A₆K)₁₅in Escherichia coli and explored its purification process. Firstly, the synthetic DNA fragment(A₆K)₁₅encoding the new surfactant-like peptide A;K was amplified and inserted into four different prokaryotic expression plasmids, pET-28 a(+), pET-28 a-CLCⅠ, pET-28 a-SUMO and pT7-GST-His. All the constructed vectors were verified by restriction enzyme digestion and sequencing. Then, the indicating vector pET28 a-CLCⅠ-(A₆K)₁₅which contain a fluorescent protein label was transformed into E. coli strain BL21(DE3), BL21(DE3) plysS and Rosetta(DE3) to find the best expression host. The IPTG concentration, induction temperature and induction time were optimized to obtain the best expression conditions. Finally, the expression product was purified by Ni-NTA chelation chromatography, and the purified(A₆K)₁₅was detected by SDS-PAGE and Western blot using the 6×His tag in(A₆K)₁₅. The product concentration was determined by the BCA method for economic benefit analysis. The results indicated that four different recombinant expression vectors containing the target gene(A₆K)₁₅were constructed. The optimal vector pET-28 a-SUMO-(A₆K)₁₅and the optimal host BL21(DE3) were screened out. The best expression condition for the surfactant-like peptide(A₆K)₁₅was IPTG 1.0 mmol/mL, induction temperature 30 ℃, and induction time 12 h. SDS-PAGE and Western blot analysis proved the molecule of Polypeptide(A₆K)₁₅. In this study, the novel surfactant-like peptide(A₆K)₁₅expression vector was successfully constructed and explored to obtain the optimal expression conditions for its large-scale preparation and purification. This research provides potential ideas for the production of new surface-active peptides by biological methods.