抗菌肽GK1在大肠杆菌中的融合表达

Fusion Expression of the Antimicrobial Peptide GK1 in Escherichia coli

摘要:

为高效表达抗菌肽GK1并避免GK1的高抗菌活性对大肠杆菌宿主菌的致命影响, 将经改造后的人胰岛素原(mhPI)与GK1的融合基因(mhPI-GK1)克隆到表达载体pET28a中, 构建出表达质粒pET28a-mhPI-GK1, 转化至大肠杆菌BL21(DE3)中进行表达。融合蛋白在大肠杆菌中以包涵体形式表达, 表达量占菌体总蛋白的20%。经CNBr裂解、阳离子交换层析和RP-HPLC纯化后, 每升发酵液可获得5.7 mg纯度大于97%的重组GK1。质谱检测显示重组GK1的分子量为2794.0 D, 抑菌活性实验表明纯化后的重组GK1和化学合成GK1具有相同的抗菌活性。为利用基因工程方法大规模生产GK1奠定了基础。

关键词:抗菌肽, 融合蛋白, 大肠杆菌

Abstract:

The antimicrobial peptide GK1, a derivative of Cecropin A, shows high antimicrobial activities against Gram-positive and Gram-negative bacteria. To avoid the lethal effects on Escherichia coli host cells during its expression, the GK1 gene was complexed with a modified human proinsulin (mhPI) gene, inserted into the vector pET28a to construct?the recombinant expression plasmid (pET28a-mhPI-GK1) and then transformed into BL21(DE3). A fusion protein was expressed and resulted in insoluble inclusion bodies counting 20% (W/W) of total cellular proteins. Recombinant GK1 was cleaved from the mhPI-GK1 fusion protein by cyanogen bromide (CNBr) and purified by cation exchange chromatography and reverse-phase HPLC. The final concentration of recombinant GK1 was 5.7 mg/L Escherichia coli culture and the purity was above 97%. Its molecular weight measured by Electrospray Ionizsation Mass Spectrometry (ESI-MS) was 2794 D, similar to that of the synthetic GK1. In addition, they have similar antibacterial activity. The results demonstrated that mhPI was capable of mediating high-level GK1 gene expression.

Key words:antimicrobial peptide, fusion protein, Escherichia coli