厚壳贻贝Mytilin-1成熟肽在毕赤酵母中的重组表达及其抑菌活性

Recombinant Expression of Mytilus coruscus Mytilin-1 Mature Peptide in Pichia pastoris and Its Antibacterial Activity

贻贝素(Mytilins)是一类主要在贝类血细胞中表达的阳离子小分子抗菌肽,包括多种同工型,它们在免疫系统中发挥了重要作用,Mytilin-1是其中的一种同工型。厚壳贻贝(Mytilus coruscus)Mytilin-1的一级结构由信号肽、成熟肽和C端的延伸肽组成;其成熟肽决定了Mytilin-1的生物学活性,它由34个氨基酸残基组成,其中8个保守的半胱氨酸残基在空间上能形成4对二硫键,对Mytilin-1的稳定性和活性起了关键作用。以厚壳贻贝的Mytilin-1成熟肽为重组DNA表达的目的蛋白,根据毕赤酵母(Pichia pastoris)的密码子偏爱性对编码该成熟肽的密码子进行优化,合成的目的基因“mMy1”与表达载体pPICZαA连接后电转至毕赤酵母X-33,通过高浓度博来霉素筛选高拷贝酵母转化子,获得的阳性转化子在29℃、250 r/min、pH6.0的条件下,使用1%甲醇诱导表达96 h;培养液上清经Tricine-SDS-PAGE和Western blot分析证明:重组mMy1在毕赤酵母X-33中得到成功表达,其分子量为4.8 kD。抑菌试验表明,重组mMy1对革兰氏阳性的金黄色葡萄球菌(Staphylococcus aureus)和枯草芽孢杆菌(Bacillus subtilis)以及革兰氏阴性的大肠杆菌(Escherchia coli)具有抑菌活性。

Mytilins are small cationic antibacterial peptides expressed predominantly in the haemocytes of shellfish,including multiple isoforms,and they play an important role in the innate immune system of shellfish.Mytilin-1 is one of the isoforms.The primary structure of Mytilus coruscus mytilin-1 is composed of signal peptide,mature peptide and C-terminal extension peptide.The mytilin-1’s mature peptide consisting of 34 amino acid residues determines its biological activity;8 conserved cysteine residues in the mature peptide may form 4 pairs of disulfide bond to stabilize the structure of mytilin-1 mature peptide.In the present study,having mytilin-1 mature peptide from M.coruscus as the recombinant DNA-expressed protein,a codon-optimized sequence(named as“mMy1”)corresponding to the cDNA encoding mytilin-1 mature peptide was synthesized based on the preferential codon usage of Pichia pastoris,and ligated to the expression vector pPICZαA and electronically transformed into competent P.pastoris X-33.The positive transformants containing multi-copy gene insertions were screened using high-concentration zeocin.The recombinant mMy1 was induced for 96 h with 1.0%methanol at 29℃,250 r/min and pH6.0.Tricine-SDS-PAGE and Western blot analysis demonstrated that the recombinant mMy1 with a molecular mass of 4.8 kD was expressed successfully in P.pastoris X-33.Bacteriostatic test indicated that the culture medium supernatant containing recombinant mMy1 showed antibacterial activity against Gram-positive(i.e.,Staphylococcus aureus and Bacillus subtilis)and Gram-negative(i.e.,Escherchia coli)bacteria.