Identification of beta-Lactamase Inhibitory Peptide Using Yeast Two-Hybrid System

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Wei Sun^1*, Youjia Hu², Jiawei Gong², Chunbao Zhu², and Baoquan Zhu²

¹School of Medicine, Tongji University, 1239 Siping Road, Shanghai 200092, China; E-mail: wayofsun1@yahoo.com.cn

²Department of Bio-Pharmaceutics, Shanghai Institute of Pharmaceutical Industry, 1320 West Beijing Road, Shanghai 200040, China

^* To whom correspondence should be addressed.

Received February 12, 2005; Revision received February 25, 2005

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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 beta-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 beta-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 beta-lactamase in vitro indicated that only P1 has the ability to inhibit beta-lactamase.

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KEY WORDS: beta-lactamase, yeast two-hybrid system, beta-lactamase inhibitory peptide, GST-peptide fusion system

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