Expression, Purification and Characterization of a Novel Hybrid Peptide CLP with Excellent Antibacterial Activity.
Expression, Purification and Characterization of a Novel Hybrid Peptide CLP with Excellent Antibacterial Activity.
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DOI：
10.3390/molecules26237142
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发表时间：
2021-11-25
期刊：
影响因子：
--
通讯作者：
Zhang R
中科院分区：
文献类型：
--
作者：
Cheng J;Ahmat M;Guo H;Wei X;Zhang L;Cheng Q;Zhang J;Wang J;Si D;Zhang Y;Zhang R
CLP是由CM 4、LL 37和TP 5合成的一种新型杂合肽，与亲本抗菌肽相比，具有显著降低的溶血活性和增加的抗菌活性。为了避免CLP的宿主毒性，获得高水平的CLP生物产量，我们在大肠杆菌中建立了His标签SUMO融合表达系统。融合蛋白可以使用镍柱纯化，通过TEV蛋白酶切割，并在镍柱的流通中进一步纯化。结果表明，重组CLP的产量为27.56 mg/L，纯度为93.6%。纯化的CLP对革兰氏阳性菌和革兰氏阴性菌具有较强的抗菌活性.碘化丙啶染色和扫描电镜观察结果表明，CLP可诱导产肠毒素大肠杆菌（ETEC）K88细胞膜透化和细胞死亡。热稳定性分析结果表明，CLP在低于70 °C的温度下保温30 min，抗菌活性略有下降，但当温度高于70 °C时，抗菌活性明显下降。此外，CLP的抑菌活性在pH 4.0 ~ 9.0范围内稳定，但pH低于4.0和高于9.0时，CLP的抑菌活性显著下降。在胃蛋白酶、木瓜蛋白酶、胰蛋白酶和蛋白酶K等多种蛋白酶存在下，CLP的抑菌活性仍保持在46.2%以上。综上所述，本研究不仅为抗菌肽的高水平生产提供了有效的策略，并对影响杂合肽CLP生物活性的干扰因素进行了评价，同时也为进一步探索治疗多重耐药细菌感染的方法铺平了道路。
CLP is a novel hybrid peptide derived from CM4, LL37 and TP5, with significantly reduced hemolytic activity and increased antibacterial activity than parental antimicrobial peptides. To avoid host toxicity and obtain high-level bio-production of CLP, we established a His-tagged SUMO fusion expression system in Escherichia coli. The fusion protein can be purified using a Nickel column, cleaved by TEV protease, and further purified in flow-through of the Nickel column. As a result, the recombinant CLP with a yield of 27.56 mg/L and a purity of 93.6% was obtained. The purified CLP exhibits potent antimicrobial activity against gram+ and gram- bacteria. Furthermore, the result of propidium iodide staining and scanning electron microscopy (SEM) showed that CLP can induce the membrane permeabilization and cell death of Enterotoxigenic Escherichia coli (ETEC) K88. The analysis of thermal stability results showed that the antibacterial activity of CLP decreases slightly below 70 °C for 30 min. However, when the temperature was above 70 °C, the antibacterial activity was significantly decreased. In addition, the antibacterial activity of CLP was stable in the pH range from 4.0 to 9.0; however, when pH was below 4.0 and over 9.0, the activity of CLP decreased significantly. In the presence of various proteases, such as pepsin, papain, trypsin and proteinase K, the antibacterial activity of CLP remained above 46.2%. In summary, this study not only provides an effective strategy for high-level production of antimicrobial peptides and evaluates the interference factors that affect the biological activity of hybrid peptide CLP, but also paves the way for further exploration of the treatment of multidrug-resistant bacterial infections.
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DOI：
10.1126/science.aau5480
发表时间：
2020-05-01
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Lazzaro BP;Zasloff M;Rolff J
通讯作者：
Rolff J
DOI：
10.3390/molecules22122071
发表时间：
2017-12-13
期刊：
Molecules (Basel, Switzerland)
影响因子：
--
作者：
Pantic JM;Jovanovic IP;Radosavljevic GD;Arsenijevic NN;Conlon JM;Lukic ML
通讯作者：
Lukic ML
影响因子：
56.9
作者：
GOLDSTEIN, G;SCHEID, MP;VANWAUWE, J
通讯作者：
VANWAUWE, J
DOI：
10.3390/molecules25235538
发表时间：
2020-11-26
期刊：
Molecules (Basel, Switzerland)
影响因子：
--
作者：
Li Z;Cheng Q;Guo H;Zhang R;Si D
通讯作者：
Si D
影响因子：
3.6
作者：
LOW, AG
通讯作者：
LOW, AG