一种新型贻贝防御素的毕赤酵母重组表达

Recombinant expression of a novel Mytilus defensin in Pichia pastoris

摘要:

贻贝含有丰富的抗菌肽(antimicrobial peptides, AMPs)，其在贻贝的先天免疫过程中发挥关键作用。贻贝抗菌肽序列较短，含多个二硫键且碱性氨基酸含量高，因此，其在原核系统(如大肠杆菌)中的功能性异源表达仍是一种挑战。为构建贻贝抗菌肽的真核重组表达体系，获得重组贻贝抗菌肽，本研究利用真核宿主毕赤酵母(Pichia pastoris)重组表达具有抑菌功能的贻贝抗菌肽myticofensin-B1，该新型防御素样抗菌肽此前已在厚壳贻贝(Mytilus coruscus)中得以鉴定。编码myticofensin-B1成熟肽段(65个氨基酸，含6个保守半胱氨酸)的基因序列经密码子优化后，克隆至pPICZαA载体，并转化至毕赤酵母GS115菌株中实现异源表达。通过液相色谱-串联质谱(liquid chromatography-tandem mass spectrometry, LC-MS/MS)证实了重组肽的序列准确性和完整性；重组肽的分子量为8 849.9 Da，与理论预测值一致。功能验证实验表明，重组myticofensin-B1具有广谱抗菌活性，对革兰氏阴性菌表现出相对更强的抑制作用。扫描电子显微镜观察发现，重组myticofensin-B1对不同细菌具有不同的作用效果。此外，重组myticofensin-B1对绵羊红细胞的溶血活性极低，但对人A549肺癌细胞表现出毒性。本研究建立了基于毕赤酵母的贻贝抗菌肽重组表达平台，表达的重组myticofensin-B1具备作为抗感染治疗药物的潜力。

Abstract:

Mytilus contain abundant antimicrobial peptides (AMPs) that play a key role in the innate immunity. However, heterologous production of these AMPs remains challenging due to their short sequences, multiple disulfide bonds, and high content of cationic amino acids, which hinder functional expression in prokaryotic systems such as Escherichia coli. To establish a eukaryotic recombinant expression system for the AMPs of mussel and obtain recombinant mussel AMPs for subsequent studies, we reported the successful recombinant expression of myticofensin-B1, a novel defensin-like AMP identified previously in Mytiluscoruscus, using the eukaryotic host Pichia pastoris. The codon-optimized gene encoding the mature myticofensin-B1 (composed of 65 amino acid residues, including 6 conserved cysteine residues) was cloned into a pPICZαA vector and expressed in P. pastoris GS115. Structural fidelity of the recombinant peptide was confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), showing a molecular weight of 8 849.9 Da, which was consistent with the theoretical prediction. Functional assays demonstrated a broad-spectrum antimicrobial activity of the recombinant myticofensin-B1, with stronger inhibition against Gram-negative bacteria. Scanning electron microscopy revealed different effects of the recombinant myticofensin-B1 against different bacteria. In addition, the recombinant myticofensin-B1 exhibited a very low hemolytic activity against sheep red blood cells and weak cytotoxicity against human A549 lung cancer cells. This study establishes P. pastoris as a powerful platform to produce functional mussel AMP and highlights the potential of the recombinant myticofensin-B1 as a therapeutic agent for aquaculture pathogens and infections.