Laboratory of Feed Biotechnology, State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China.
Molecules. 2018 Jun 20;23(6):1491. doi: 10.3390/molecules23061491.
The hybrid peptide cecropin A (1⁻8)⁻LL37 (17⁻30) (C⁻L), derived from the sequence of cecropin A (C) and LL-37 (L), showed significantly increased antibacterial activity and minimized hemolytic activity than C and L alone. To obtain high-level production of C⁻L, the deoxyribonucleic acid sequence encoding C⁻L with preferred codons was cloned into pET-SUMO to construct a fusion expression vector, and overexpressed in () BL21 (DE3). The maximum fusion protein (92% purity) was obtained with the yield of 89.14 mg/L fermentation culture after purification with Ni-NTA Sepharose column. The hybrid C⁻L was cleaved from the fusion protein by SUMO-protease, and 17.54 mg/L pure active C⁻L was obtained. Furthermore, the purified C⁻L showed identical antibacterial and hemolytic activity to synthesized C⁻L. Stability analysis results exhibited that the activity of C⁻L changed little below 80 °C for 20 min, but when the temperature exceeded 80 °C, a significant decrease was observed. Varying the pH from 5.0 to 10.0 did not appear to influence the activity of C⁻L, however, pH below 4.0 decreased the antibacterial activity of C⁻L rapidly. Under the challenge of several proteases (pepsin, trypsin, and proteinase K), the functional activity of C⁻L was maintained over 50%. In summary, this study not only supplied an effective approach for high-level production of hybrid peptide C⁻L, but paved the way for its further exploration in controlling infectious diseases of farm animals or even humans.
由抗菌肽蜂毒素 A (1⁻8)⁻LL37 (17⁻30)（C⁻L）的序列衍生而来的融合肽，与 C 和 L 相比，显示出显著增强的抗菌活性和最小化的溶血活性。为了获得 C⁻L 的高水平生产，用优选密码子对编码 C⁻L 的脱氧核糖核酸序列进行克隆，构建融合表达载体，并在 () BL21 (DE3) 中过表达。经过 Ni-NTA Sepharose 柱纯化，获得了最大纯度为 92%的融合蛋白（产量为 89.14 mg/L 发酵液）。融合蛋白用 SUMO 蛋白酶切割后，得到 17.54 mg/L 的纯活性 C⁻L。此外，纯化的 C⁻L 显示出与合成的 C⁻L 相同的抗菌和溶血活性。稳定性分析结果表明，C⁻L 的活性在 80°C 以下 20 分钟内变化不大，但当温度超过 80°C 时，活性显著下降。C⁻L 的活性在 pH5.0 到 10.0 之间变化时似乎没有影响，但 pH 低于 4.0 时，C⁻L 的抗菌活性迅速下降。在几种蛋白酶（胃蛋白酶、胰蛋白酶和蛋白酶 K）的挑战下，C⁻L 的功能活性保持在 50%以上。总之，本研究不仅为高效生产融合肽 C⁻L 提供了一种有效的方法，而且为其在控制农场动物甚至人类传染病方面的进一步探索铺平了道路。
