Expression and Purification of Hybrid LL-37Tα1 Peptide in Pichia pastoris and Evaluation of Its Immunomodulatory and Anti-inflammatory Activities by LPS Neutralization
Expression and Purification of Hybrid LL-37Tα1 Peptide in Pichia pastoris and Evaluation of Its Immunomodulatory and Anti-inflammatory Activities by LPS Neutralization
复制标题
杂交 LL-37T α1 肽在毕赤酵母中的表达和纯化及其通过 LPS 中和评价其免疫调节和抗炎活性
DOI：
10.3389/fimmu.2019.01365
复制
发表时间：
2019-06-14
影响因子：
7.3
通讯作者：
Zhang Rijun
中科院分区：
文献类型：
--
作者：
Ahmad, Baseer;Hanif, Quratulain;Zhang Rijun
本研究涉及杂交肽LL-37 T α 1的开发及其生物医学应用的新方法。线性阳离子杂合肽LL-37 T α 1衍生自两种亲本肽（LL-37和T α 1），其被认为是有效的抗内毒素而没有任何溶血或细胞毒性活性。我们成功地将杂合肽LL-37 T α 1基因克隆到PpICZ α A载体中，并在毕赤酵母中表达。重组肽经镍亲和柱和反相高效液相色谱（RP-HPLC）纯化，SDS-PAGE和质谱测定其相对分子质量为3.9 kDa。我们分析了鲎变形细胞溶解物（LAL）活性对LPS的中和作用，结果表明杂合肽LL-37 T α 1直接结合内毒素，并以剂量依赖性方式显著（p < 0.05）中和LPS的作用。乳酸脱氢酶（LDH）测定显示LL-37 T α 1成功地降低了小鼠RAW 264.7巨噬细胞中LPS诱导的细胞毒性。此外，它显著（p < 0.05）降低了LPS刺激的小鼠RAW 264.7巨噬细胞中一氧化氮、促炎细胞因子（包括TNF-α、IL-6、IL-1 β）的水平，并减少了凋亡细胞的数量。我们的研究结果表明，巴斯德毕赤酵母表达系统是具有成本效益的商业生产的免疫调节和抗炎杂合肽（IAHP）LL-37 T α 1和肽可以作为有效的抗内毒素/抗炎剂与最小的细胞毒性。
This study pertains to the new approach for the development of hybrid peptide LL-37T alpha 1 and its biomedical applications. A linear cationic hybrid peptide, LL-37T alpha 1 was derived from two parental peptides (LL-37 and T alpha 1) recognized as potent anti-endotoxin without any hemolytic or cytotoxic activity. We successfully cloned the gene of hybrid peptide LL-37T alpha 1 in PpICZ alpha A vector and expressed in the Pichia pastoris. The recombinant peptide was purified by Ni-affinity column and reverse-phase high performance liquid chromatography (RP-HPLC) with an estimated molecular mass of 3.9 kDa as determined by SDS-PAGE and mass spectrometry. We analyzed the LPS neutralization by limulus amebocyte lysate (LAL) activity and the results indicate that the hybrid peptide LL-37T alpha 1 directly binds endotoxin and significantly (p < 0.05) neutralizes the effect of LPS in a dose-dependent manner. Lactate dehydrogenase (LDH) assay revealed that LL-37T alpha 1 successfully reduces the LPS-induced cytotoxicity in mouse RAW264.7 macrophages. Moreover, it significantly (p < 0.05) decreased the levels of nitric oxide, proinflammatory cytokines including TNF-alpha, IL-6, IL-1 beta, and diminished the number of apoptotic cells in LPS-stimulated mouse RAW264.7 macrophages. Our results suggest that the P pastoris expression system is cost-effective for commercial production of the immunomodulatory and anti-inflammatory hybrid peptide (IAHP) LL-37T alpha 1 and the peptide may serve as effective anti-endotoxin/anti-inflammatory agent with minimal cytotoxicity.