Identification of beta-lactamase inhibitory peptide using yeast two-hybrid system.
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.
Sun
Wei
W
School of Medicine, Tongji University, Shanghai, 200092, China. wayofsun1@yahoo.com.cn
Hu
Youjia
Y
Gong
Jiawei
J
Zhu
Chunbao
C
Zhu
Baoquan
B
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D015195:Drug Design; D004353:Drug Evaluation, Preclinical; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D019151:Peptide Library; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D013329:Structure-Activity Relationship; D020798:Two-Hybrid System Techniques; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism
D004791:Enzyme Inhibitors; D010446:Peptide Fragments; D019151:Peptide Library; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases