Identification of β-Lactamase Inhibitory Peptide Using Yeast Two-Hybrid System
Identification of β-Lactamase Inhibitory Peptide Using Yeast Two-Hybrid System
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DOI：
10.1007/s10541-005-0180-6
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发表时间：
2005-07
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影响因子：
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通讯作者：
Wei Sun;Youjia Hu;Jiawei Gong;Chunbao Zhu;Baoquan Zhu
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文献类型：
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作者：
Wei Sun;Youjia Hu;Jiawei Gong;Chunbao Zhu;Baoquan Zhu
设计随机寡核苷酸片段，经聚合酶链式反应扩增，与pGAD424的活化区融合，构建随机多肽文库。将编码β-内酰胺酶的基因片段与pGBT9(+2)的结合区进行融合。随后，利用酵母双杂交系统，我们发现了两个编码多肽P1和P2的阳性克隆，它们在体内具有结合β-内酰胺酶的能力。将编码P1和P2的基因克隆到pGEX-4T-1中。在大肠杆菌中表达了GST-肽融合蛋白，并用谷胱甘肽-Sepharose4B亲和层析进行了分离。最后，用凝血酶从融合蛋白中切割出P1和P2，并通过超滤纯化。β-内酰胺酶体外抑制试验表明，只有P1对β-内酰胺酶有抑制作用。
Random oligonucleotide fragments were designed and amplified by PCR and fused with the activating domain of pGAD424 to construct a random peptide library. The DNA fragment encoding β-lactamase was fused with the binding domain of pGBT9 (+2). Subsequently, using yeast two-hybrid system we found two positive clones encoding peptides P1 and P2 that have the ability to bind β-lactamasein vivo. The genes encoding P1 and P2 were cloned into pGEX-4T-1. GST-peptide fusion proteins were expressed inEscherichia coliand isolated by glutathione-Sepharose 4B affinity chromatography. Finally, P1 and P2 were cleaved from the fusion protein with thrombin and purified by ultrafiltration. Inhibition assay of peptides with β-lactamasein vitroindicated that only P1 has the ability to inhibit β-lactamase.