ABSTRACT
We investigated the application of resins used in solid-phase synthesis for affinity purification. A synthetic ligand for FK506-binding protein 12 (SLF) was immobilized on various resins, and the binding assays between the SLF-immobilized resins and FK506-binding protein 12 (FKBP12) were performed. Of the resins tested in this study, PEGA resin was the most effective for isolating FKBP12. This matrix enabled the isolation of FKBP12 from a cell lysate, and the identification of SLF-binding peptides from a phage cDNA library. We confirmed the interaction between SLF and these peptides using a cuvette type quartz crystal microbalance (QCM) apparatus. Our study suggests that PEGA resin has great potential as a tool not only for the purification and identification of small-molecule binding proteins but also for the selection of peptides that recognize target molecules.
Subject(s)
Acrylic Resins/chemistry , Alkanes/chemistry , Alkanes/metabolism , Piperidines/chemistry , Piperidines/metabolism , Polyethylene Glycols/chemistry , Tacrolimus Binding Protein 1A/analysis , Tacrolimus Binding Protein 1A/metabolism , Amino Acid Sequence , Bacteriophage T7/genetics , Bacteriophage T7/metabolism , Cloning, Molecular , Gene Library , Humans , Jurkat Cells , Kinetics , Ligands , Molecular Sequence Data , Peptides/analysis , Peptides/chemistry , Peptides/metabolism , Protein Binding , Substrate Specificity , Tacrolimus Binding Protein 1A/isolation & purificationABSTRACT
Podophyllotoxin (PT), a strong cytotoxic agent from berberidaceae, has been known to inhibit tubulin polymerization. Although PT has been used for developing anticancer drugs as one of seed compounds, clinical treatment by itself has been unsuccessful because of the side effects, except one example in the treatments of warts. In this study, we screened peptides binding to PT with T7 phage display clonings in order to obtain more information about molecular mechanism of the action. A selected phage clone has a specific amino acid sequence to be SVPSRRRPDGRTHRSSRG. A homology search by protein database BLAST showed that this sequence had a similarity to a hinge domain (HD) of E2 protein in human papillomavirus (HPV) type 1a which is known to cause plantar warts. Surface plasmon resonance (SPR) analysis showed that PT bound to a recombinant HPV 1a E2 protein giving a K(D)=24.1microM which has compared with those of other domains of E2 protein. Also we demonstrated whether PT inhibited HD interaction or not. E7 protein of HPV has been known to be an oncoprotein and was reported to interact with HD of E2 protein. We demonstrated that an E2/E7 interaction was inhibited by the addition of PT in this report. And we showed the bindings of PT to other types of HPV. Our results suggest that PT is potential as a tool for clarifying the molecular mechanism of HPV.
Subject(s)
DNA-Binding Proteins/chemistry , DNA-Binding Proteins/drug effects , Oncogene Proteins, Viral/chemistry , Oncogene Proteins, Viral/drug effects , Podophyllotoxin/pharmacology , Animals , Binding Sites , Cell Line, Tumor , Cell Proliferation/drug effects , DNA-Binding Proteins/genetics , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical , Mice , Molecular Conformation , Oncogene Proteins, Viral/genetics , Papillomavirus E7 Proteins , Peptide Library , Podophyllotoxin/chemical synthesis , Podophyllotoxin/chemistry , Protein Binding/drug effects , Protein Structure, Tertiary/drug effects , Stereoisomerism , Structure-Activity Relationship , Time FactorsABSTRACT
Peptide libraries displayed by T7 phage, which contain random cDNA fragments insets, were screened for their ability to bind to a biotinylated derivative of clarithromycin. Phage particles bound to an immobilized derivative of the antibiotic were isolated and the inserted cDNA was amplified and sequenced. A common selected peptide sequence, composed of 19 amino acids, was obtained and a synthetic peptide with this sequence was produced. Surface plasmon resonance experiments showed that the synthetic peptide immobilized on a sensor chip bound to clarithromycin and the dissociation constant was determined to be 2.1 x 10(-3) M. The dissociation constants of other macrolide antibiotics, erythromycin, roxithromycin, azithromycin and josamycin were also determined to be 5.4 x 10(-3) M, 6.2 x 10(-5) M, 1.1 M and 3.4 x 10(-2) M, respectively. These results indicated that T7 phage display method might be useful to determine relatively weak interactions between small molecule drugs and the selected peptides which could represent a possible binding site conserved in binding proteins.
