The purpose of this study was to screen peptides that can specifically bind to human hepatocellular carcinoma (hHCC) cells using phage display of random peptide library in order to develope a peptide-based carrier f...The purpose of this study was to screen peptides that can specifically bind to human hepatocellular carcinoma (hHCC) cells using phage display of random peptide library in order to develope a peptide-based carrier for the diagnosis or therapy of hHCC. A peptide 12-mer phage display library was employed and 4 rounds of subtractive panning were performed using the hHCC cell line HepG2 as the target. After panning, the phages that specifically bound to and internalized in hHCC cells were selected. The selected phages demonstrated highly specific affinity to HepG2 cells analyzed by ELISA and immunofluorescence analysis. 57.3% of the selected phage clones displayed repeated sequence FLLEPHLMDTSM, and 4 amino acid residues, FLEP were extremely conservative. Based on the sequencing results, a 16-mer peptide (WH-16) was synthesized. The competitive EL1SA showed that the binding of the phage clones displayed sequence FLLEPHLMDTSM to HepG2 cells was efficiently inhibited by WH-16. Our findings indicate that cellular binding of phage is mediated via its displayed peptide and the synthesized 16-mer peptide may have the potential to be a delivery carrier in target diagnosis or therapy for hHCC.展开更多
Endoglucanases are the main cellulolytic enzymes digestion as well as its good kinetic properties make it an attractive of Anoplophora glabripennis. Their high activities in cellulose target for development of cellula...Endoglucanases are the main cellulolytic enzymes digestion as well as its good kinetic properties make it an attractive of Anoplophora glabripennis. Their high activities in cellulose target for development of cellulase inhibitors. In this study, random pepfide phage display technology was employed to identify peptides that bound the AgEG1, a member of endoglucanase isozymes. Phage clones with peptide LPPNPTK and XPP (X is residue T, L, A or H) motif frequently occurred in the selected phage population and showed a higher phage recovery than other clones. Peptide LPPNPTK was chemically synthesized and characterized tor its binding activities to AgEG1. The synthetic peptide exhibited high specificity for AgEG1. The peptide LPPNPTK has the potential to be developed into inhibitors of the endoglucanase of A. glabripennis.展开更多
Tumor necrosis factor(TNF)-α-converting enzyme(TACE)is the major protease responsible for processing pro-TNF-αfrom membrane-anchored precursors to secreted TNF-α.In the present study,a 15-peptide library was used t...Tumor necrosis factor(TNF)-α-converting enzyme(TACE)is the major protease responsible for processing pro-TNF-αfrom membrane-anchored precursors to secreted TNF-α.In the present study,a 15-peptide library was used to identify potential TACE antagonists.To obtain the recombinant TACE ectodomain and to use it as a selective molecule for the screening of peptide inhibitors of TACE,cDNA coding for the catalytic domain(T800)and full-length ectodomain(T1300)of TACE were amplified by reverse transcription–polymerase chain reaction.The expression plasmid were constructed by inserting T800/T1300 into plasmid pET-28a/c respectively and were transformed into Escherichia coli BL21(DE3).Sodium dodecyl sulfate–polyacrylamide gel electrophoresis(SDSPAGE)andWestern blot analysis revealed that T800/T1300 were highly expressed in the form of an inclusion body induced by isopropylthiogalactoside.After Ni2+–NTA resin affinity chromatography,the purity of the recombinant T800/T1300 protein was more than 90%.T800 and T1300 proteins were used in the screening of T800/T1300-binding peptides from a phage display random 15-peptide library.After four rounds of biopanning,the positive phage clones were analyzed by enzyme-linked immunosorbent assay,competitive inhibition assay(ELESA),and DNA sequencing.A common amino acid sequence(TRWLVYFS RPYLVAT)was confirmed and synthesized.A synthetic peptide was shown to bind to TACE and to inhibit TNF-αrelease from lipopolysaccharide(LPS)-stimulated human peripheral blood mononuclear cells(PBMC)by up to 60.3%.Fluorescence-activated cell sorter(FACS)analysis revealed that the peptide mediated the accumulation of TNF-αon an LPS-stimulated PBMC surface.These results demonstrate that the TACE-binding peptide is an effective antagonist of TACE and that the deduced motif might be applied to the molecular design of anti-inflammatory drugs.展开更多
To develop a targeting vector for breast cancer biotherapy, MDA-MB-231 cell, a human breast cancer cell line, was co-cultured with pC89 (9 aa) phage display library of random peptides. In multiple inde- pendent peptid...To develop a targeting vector for breast cancer biotherapy, MDA-MB-231 cell, a human breast cancer cell line, was co-cultured with pC89 (9 aa) phage display library of random peptides. In multiple inde- pendent peptide-presenting phage screening trials, subtilisin was used as a protease to inactivate ex- tra-cellular phages. The internalized phages were collected by cell lysising and amplified in E. coli XLI-Blue. Through five rounds of selection, the peptide-presenting phages which could be internalized in MDA-MB-231 cells were isolated. A comparison was made between internalization capacities of pep- tide-presenting phages isolated from MDA-MB-231 cells and RGD-integrin binding phage by cocultur- ing them with other human tumor cell lines and normal cells. The nucleotide sequences of isolated peptide-presenting phages were then determined by DNA sequencing. To uncover whether phage coat protein or amino acid order was required for the character of the peptide to MDA-MB-231 cells, three peptides were synthesized. They are CASPSGALRSC, ASPSGALRS and CGVIFDHSVPC (the shifted sequence of CASPSGALRSC), and after coculturing them with different cell lines, their targeting ca- pacities to MDA-MB-231 cells were detected. These data suggested that the internalization process was highly selective, and capable of capturing a specific peptide from parent peptide variants. Moreover, the targeting internalization event of peptides was an amino acid sequence dependent manner. The results demonstrated the feasibility of using phage display library of random peptides to develop new targeting system for intracellular delivery of macromolecules, and the peptide we obtained might be modified as a targeting vector for breast cancer gene therapy.展开更多
Brain-derived neurotrophic factor (BDNF) shows potential in the treatment of neurode-generative diseases, but the therapeutic application of BDNF has been greatly limited because it is too large in molecular size to p...Brain-derived neurotrophic factor (BDNF) shows potential in the treatment of neurode-generative diseases, but the therapeutic application of BDNF has been greatly limited because it is too large in molecular size to permeate blood-brain barrier. To develop low-molecular-weight BDNF-like peptides, we selected a phage-displayed random peptide library using trkB expressed on NIH 3T3 cells as target in the study. With the strategy of peptide library incubation with NIH 3T3 cells and competitive elution with 1 mg/mL of BDNF in the last round of selection, the specific phages able to bind to the natural conformation of trkB and antagonize BDNF binding to trkB were enriched effectively. Five trkB-binding peptides were obtained, in which a core sequence of CRA/TXfXXfXXC (X represents the random amino acids, f represents T, L or I) was identified. The BDNF-like activity of these five peptides displayed on phages was not observed, though all of them antagonized the activity of BDNF in a dose-dependent manner. Similar results were obtained with the synthetic peptide of C1 clone, indicating that the 5 phage-derived peptides were trkB antagonists. These low-molecular-weight antagonists of trkB may be of potential application in the treatment of neuroblastoma and chronic pain. Meanwhile, the obtained core sequence also could be used as the base to construct the secondary phage-displayed peptide library for further devel-opment of small peptides mimicking BDNF activity.展开更多
Analysis of proteins that interact with N protein of SARS-CoV using 15-mer phage-displayed library will help to explore the virus pathogenesis and to develop new drugs and vaccines against SARS. In this study,we clone...Analysis of proteins that interact with N protein of SARS-CoV using 15-mer phage-displayed library will help to explore the virus pathogenesis and to develop new drugs and vaccines against SARS. In this study,we cloned,expressed and purified N protein of SARS-CoV. This 46-kD N protein was verified by SDS-PAGE and Western-blot. Then,the peptides binding-specific to N protein were identified using 15-mer phage-displayed library. Surprisingly,all of the 89 clones from monoclonal ELISA were positive (S/N>2.1) and the result was further confirmed experimentally once again. Six N protein-binding pep-tides,designated separately as SNA1,SNA2,SNA4,SNA5,SNA9 and SNG11,were selected for se-quencing. Sequence analysis suggested that SNA5 shared approximatively 100% sequence identity to SNA4,SNA2,SNA9 and SNA1. In addition,the binding specificity of the 15-mer peptides with the SARS-CoV N protein was further demonstrated by blocking ELISA using the synthetical 15-mer peptide according to the deduced amino acid sequence of SNA5. Also,the deduced amino sequence of SNA5 was compared with proteins in translated database using the tblastx program,and the results showed that the proteins with the highest homology were Ubiquinol-cytochrome c reductase iron-sulfur sub-units (UCRI or UQCR),otherwise known as the Rieske iron-sulfur proteins (RISP). Notablely,in the 2Fe-2S redox centre of UCRI,there were 6 residues GGW(Y)F(Y)CP compatible to the residues (po-sition 2→7,GGWFCP7) of the NH2-terminal of the 15-mer peptide,which indicated higher binding specificity between the N protein of SARS-CoV and the redox centre of UCRI to some extent. Here,the possible molecular mechanisms of SARS-CoV N protein in the pathogenesis of SARS are discussed.展开更多
To obtain short peptides simulating antigenic epitopes related to natural resistance against Schistosoma japonicum (S.j) in rats, and to explore their immune protection against S.j in mice, phage random peptide librar...To obtain short peptides simulating antigenic epitopes related to natural resistance against Schistosoma japonicum (S.j) in rats, and to explore their immune protection against S.j in mice, phage random peptide library of 12 amino acids were screened with purified IgG from normal rat sera. Positive clones that were obtained after three rounds of biopanning were detected by ELISA, and two of them were sequenced. Kunming mice were immunized with mixed phage clones. Each mouse was challenged with 40±1 S.j cercariae, and all mice were perfused 45 days post-challenge. The worms and the liver eggs were counted. The results were that the specific phages binding to IgG were enriched 300 folds after three rounds of biopanning. Twenty clones were detected by ELISA and 19 of them bound to the specific IgG of rat sera. The sequence of two clones revealed no homology with other sequences in the GenBank. Compared with the control groups, the reduction rates of the worm burden and liver egg were 33.6% and 59.8%, respectively. It was concluded that the specific peptides, which simulate antigenic molecules correlated with natural resistance to S.j in rats could be obtained by immunosceening phage random peptide library and a protective immunity against S.j can be detected by these epitopes in mice.展开更多
基金a grant from National Natural Science Foundation of China(N0.30171062)
文摘The purpose of this study was to screen peptides that can specifically bind to human hepatocellular carcinoma (hHCC) cells using phage display of random peptide library in order to develope a peptide-based carrier for the diagnosis or therapy of hHCC. A peptide 12-mer phage display library was employed and 4 rounds of subtractive panning were performed using the hHCC cell line HepG2 as the target. After panning, the phages that specifically bound to and internalized in hHCC cells were selected. The selected phages demonstrated highly specific affinity to HepG2 cells analyzed by ELISA and immunofluorescence analysis. 57.3% of the selected phage clones displayed repeated sequence FLLEPHLMDTSM, and 4 amino acid residues, FLEP were extremely conservative. Based on the sequencing results, a 16-mer peptide (WH-16) was synthesized. The competitive EL1SA showed that the binding of the phage clones displayed sequence FLLEPHLMDTSM to HepG2 cells was efficiently inhibited by WH-16. Our findings indicate that cellular binding of phage is mediated via its displayed peptide and the synthesized 16-mer peptide may have the potential to be a delivery carrier in target diagnosis or therapy for hHCC.
基金Supported by the National Natural Science Foundation of China (Grant No. 39900116)
文摘Endoglucanases are the main cellulolytic enzymes digestion as well as its good kinetic properties make it an attractive of Anoplophora glabripennis. Their high activities in cellulose target for development of cellulase inhibitors. In this study, random pepfide phage display technology was employed to identify peptides that bound the AgEG1, a member of endoglucanase isozymes. Phage clones with peptide LPPNPTK and XPP (X is residue T, L, A or H) motif frequently occurred in the selected phage population and showed a higher phage recovery than other clones. Peptide LPPNPTK was chemically synthesized and characterized tor its binding activities to AgEG1. The synthetic peptide exhibited high specificity for AgEG1. The peptide LPPNPTK has the potential to be developed into inhibitors of the endoglucanase of A. glabripennis.
基金supported by National Nature Science Foundation of China (No.30070722).
文摘Tumor necrosis factor(TNF)-α-converting enzyme(TACE)is the major protease responsible for processing pro-TNF-αfrom membrane-anchored precursors to secreted TNF-α.In the present study,a 15-peptide library was used to identify potential TACE antagonists.To obtain the recombinant TACE ectodomain and to use it as a selective molecule for the screening of peptide inhibitors of TACE,cDNA coding for the catalytic domain(T800)and full-length ectodomain(T1300)of TACE were amplified by reverse transcription–polymerase chain reaction.The expression plasmid were constructed by inserting T800/T1300 into plasmid pET-28a/c respectively and were transformed into Escherichia coli BL21(DE3).Sodium dodecyl sulfate–polyacrylamide gel electrophoresis(SDSPAGE)andWestern blot analysis revealed that T800/T1300 were highly expressed in the form of an inclusion body induced by isopropylthiogalactoside.After Ni2+–NTA resin affinity chromatography,the purity of the recombinant T800/T1300 protein was more than 90%.T800 and T1300 proteins were used in the screening of T800/T1300-binding peptides from a phage display random 15-peptide library.After four rounds of biopanning,the positive phage clones were analyzed by enzyme-linked immunosorbent assay,competitive inhibition assay(ELESA),and DNA sequencing.A common amino acid sequence(TRWLVYFS RPYLVAT)was confirmed and synthesized.A synthetic peptide was shown to bind to TACE and to inhibit TNF-αrelease from lipopolysaccharide(LPS)-stimulated human peripheral blood mononuclear cells(PBMC)by up to 60.3%.Fluorescence-activated cell sorter(FACS)analysis revealed that the peptide mediated the accumulation of TNF-αon an LPS-stimulated PBMC surface.These results demonstrate that the TACE-binding peptide is an effective antagonist of TACE and that the deduced motif might be applied to the molecular design of anti-inflammatory drugs.
基金the National Natural Science Foundation of China (Grant No. 30460142)
文摘To develop a targeting vector for breast cancer biotherapy, MDA-MB-231 cell, a human breast cancer cell line, was co-cultured with pC89 (9 aa) phage display library of random peptides. In multiple inde- pendent peptide-presenting phage screening trials, subtilisin was used as a protease to inactivate ex- tra-cellular phages. The internalized phages were collected by cell lysising and amplified in E. coli XLI-Blue. Through five rounds of selection, the peptide-presenting phages which could be internalized in MDA-MB-231 cells were isolated. A comparison was made between internalization capacities of pep- tide-presenting phages isolated from MDA-MB-231 cells and RGD-integrin binding phage by cocultur- ing them with other human tumor cell lines and normal cells. The nucleotide sequences of isolated peptide-presenting phages were then determined by DNA sequencing. To uncover whether phage coat protein or amino acid order was required for the character of the peptide to MDA-MB-231 cells, three peptides were synthesized. They are CASPSGALRSC, ASPSGALRS and CGVIFDHSVPC (the shifted sequence of CASPSGALRSC), and after coculturing them with different cell lines, their targeting ca- pacities to MDA-MB-231 cells were detected. These data suggested that the internalization process was highly selective, and capable of capturing a specific peptide from parent peptide variants. Moreover, the targeting internalization event of peptides was an amino acid sequence dependent manner. The results demonstrated the feasibility of using phage display library of random peptides to develop new targeting system for intracellular delivery of macromolecules, and the peptide we obtained might be modified as a targeting vector for breast cancer gene therapy.
基金the National Natural Science Foundation of China (Grant Nos. 39825116 & 30070168). References
文摘Brain-derived neurotrophic factor (BDNF) shows potential in the treatment of neurode-generative diseases, but the therapeutic application of BDNF has been greatly limited because it is too large in molecular size to permeate blood-brain barrier. To develop low-molecular-weight BDNF-like peptides, we selected a phage-displayed random peptide library using trkB expressed on NIH 3T3 cells as target in the study. With the strategy of peptide library incubation with NIH 3T3 cells and competitive elution with 1 mg/mL of BDNF in the last round of selection, the specific phages able to bind to the natural conformation of trkB and antagonize BDNF binding to trkB were enriched effectively. Five trkB-binding peptides were obtained, in which a core sequence of CRA/TXfXXfXXC (X represents the random amino acids, f represents T, L or I) was identified. The BDNF-like activity of these five peptides displayed on phages was not observed, though all of them antagonized the activity of BDNF in a dose-dependent manner. Similar results were obtained with the synthetic peptide of C1 clone, indicating that the 5 phage-derived peptides were trkB antagonists. These low-molecular-weight antagonists of trkB may be of potential application in the treatment of neuroblastoma and chronic pain. Meanwhile, the obtained core sequence also could be used as the base to construct the secondary phage-displayed peptide library for further devel-opment of small peptides mimicking BDNF activity.
基金the Science and Technology Project of Chongqing Municipal Educa- tion Commission, China (Grant No. KJ071109)the Key Project of the Ministry of Science and Technology of China (Grant No. 2003CB514120)
文摘Analysis of proteins that interact with N protein of SARS-CoV using 15-mer phage-displayed library will help to explore the virus pathogenesis and to develop new drugs and vaccines against SARS. In this study,we cloned,expressed and purified N protein of SARS-CoV. This 46-kD N protein was verified by SDS-PAGE and Western-blot. Then,the peptides binding-specific to N protein were identified using 15-mer phage-displayed library. Surprisingly,all of the 89 clones from monoclonal ELISA were positive (S/N>2.1) and the result was further confirmed experimentally once again. Six N protein-binding pep-tides,designated separately as SNA1,SNA2,SNA4,SNA5,SNA9 and SNG11,were selected for se-quencing. Sequence analysis suggested that SNA5 shared approximatively 100% sequence identity to SNA4,SNA2,SNA9 and SNA1. In addition,the binding specificity of the 15-mer peptides with the SARS-CoV N protein was further demonstrated by blocking ELISA using the synthetical 15-mer peptide according to the deduced amino acid sequence of SNA5. Also,the deduced amino sequence of SNA5 was compared with proteins in translated database using the tblastx program,and the results showed that the proteins with the highest homology were Ubiquinol-cytochrome c reductase iron-sulfur sub-units (UCRI or UQCR),otherwise known as the Rieske iron-sulfur proteins (RISP). Notablely,in the 2Fe-2S redox centre of UCRI,there were 6 residues GGW(Y)F(Y)CP compatible to the residues (po-sition 2→7,GGWFCP7) of the NH2-terminal of the 15-mer peptide,which indicated higher binding specificity between the N protein of SARS-CoV and the redox centre of UCRI to some extent. Here,the possible molecular mechanisms of SARS-CoV N protein in the pathogenesis of SARS are discussed.
基金This study was supported by grants from WHO/TDR (980255) and the Science Commission of Hunan Province (00jzy2115)
文摘To obtain short peptides simulating antigenic epitopes related to natural resistance against Schistosoma japonicum (S.j) in rats, and to explore their immune protection against S.j in mice, phage random peptide library of 12 amino acids were screened with purified IgG from normal rat sera. Positive clones that were obtained after three rounds of biopanning were detected by ELISA, and two of them were sequenced. Kunming mice were immunized with mixed phage clones. Each mouse was challenged with 40±1 S.j cercariae, and all mice were perfused 45 days post-challenge. The worms and the liver eggs were counted. The results were that the specific phages binding to IgG were enriched 300 folds after three rounds of biopanning. Twenty clones were detected by ELISA and 19 of them bound to the specific IgG of rat sera. The sequence of two clones revealed no homology with other sequences in the GenBank. Compared with the control groups, the reduction rates of the worm burden and liver egg were 33.6% and 59.8%, respectively. It was concluded that the specific peptides, which simulate antigenic molecules correlated with natural resistance to S.j in rats could be obtained by immunosceening phage random peptide library and a protective immunity against S.j can be detected by these epitopes in mice.