Identification of β-Lactamase Inhibitory Peptide Using Yeast Two-Hybrid System
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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-lactamase in vivo. The genes encoding P1 and P2 were cloned into pGEX-4T-1. GST-peptide fusion proteins were expressed in Escherichia coli and 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-lactamase in vitro indicated that only P1 has the ability to inhibit β-lactamase.
Key wordsβ-lactamase yeast two-hybrid system β-lactamase inhibitory peptide GST-peptide fusion system
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