• Title/Summary/Keyword: Phage display

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Identification of Dinitrotoluene Selective Peptides by Phage Display Cloning

  • Jang, Hyeon-Jun;Na, Jung-Hyun;Jin, Bong-Suk;Lee, Won-Kyu;Lee, Woong-Hee;Jung, Hyun-Jin;Kim, Seok-Chan;Lim, Si-Hyung;Yu, Yeon-Gyu
    • Bulletin of the Korean Chemical Society
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    • v.31 no.12
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    • pp.3703-3706
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    • 2010
  • Biomolecules specific to explosives can be exploited as chemical sensors. Peptides specific to immobilized dinitrotoluene (DNT) were identified using a phage display library. A derivative of DNT that contained an extended amine group, 4-(2,4-dinitrophenyl)butan-1-amine, was synthesized and immobilized using a self-assembled monolayer surface on gold. Filamentous M13 phages displaying random sequences of 12-mer peptides specific to the immobilized DNT-derivate were isolated from the M13 phage library by biopanning. A common peptide sequence was identified from the isolated phages and the synthesized peptides showed selective binding to DNT. When the peptide was immobilized on a quartz crystal microbalance (QCM) chip, it showed a binding signal to DNT, while toluene barely showed significant binding to the QCM chip. These results demonstrate that peptides screened by biopanning against immobilized DNT can be useful for quick and accurate detection of DNT.

Terminal Protein-specific scFv Production by Phage Display (Phage Display 방법을 이용한 B형 간염 바이러스의 Terminal Protein 특이 scFv 항체 생산)

  • Lee, Myung-Shin;Kwon, Myung-Hee;Park, Sun;Shin, Ho-Joon;Kim, Hyung-Il
    • IMMUNE NETWORK
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    • v.3 no.2
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    • pp.126-135
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    • 2003
  • Background: One of the important factors in the prognosis of chronic hepatitis B patient is the degree of replication of hepatitis B virus (HBV). It has been known that HBV DNA polymerase plays the essential role in the replication of HBV. HBV DNA polymerase is composed of four domains, TP (Terminal protein), spacer, RT (Reverse transcriptase) and RNaseH. Among these domains, tyrosine, the $65^{th}$ residue of TP is an important residue in protein-priming reaction that initiates reverse transcription. If monoclonal antibody that recognizes around tyrosine residue were selected, it could be applied to further study of HBV replication. Methods: To produce TP-specific scFv (single-chain Fv) by phage display, mice were immunized using synthetic TP-peptide contains $57{\sim}80^{th}$ amino acid residues of TP domain. After isolation of mRNA of heavy-variable region ($V_H$) and light-chain variable region ($V_L$) from the spleen of the immunized mouse, DNA of $V_H$ and $V_L$ were obtained by RT-PCR and joined by a DNA linker encoding peptide (Gly4Ser)3 as a scFv DNA fragments. ScFv DNA fragments were cloned into a phagemid vector. scFv was expressed in E.coli TG1 as a fusion protein with E tag and phage gIII. To select the scFv that has specific affinity to TP-peptide from the phage-antibody library, we used two cycles of panning and colony lift assay. Results: The TP-peptide-specific scFv was isolated by selection process using TP-peptide as an antigen. Selected scFv had 30 kDa of protein size and its nucleotide sequences were analyzed. Indirect- and competitive-ELISA revealed that the selected scFv specifically recognized both TP-peptide and the HBV DNA polymerase. Conclusion: The scFv that recognizes the TP domain of the HBV DNA polymerase was isolated by phage display.

Construction of a Hexapeptide Library using Phage Display for Bio-panning

  • Cho, Won-Hee;Yoo, Seung-Ku
    • Journal of Microbiology
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    • v.37 no.2
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    • pp.97-101
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    • 1999
  • Random hexapeptide library on the surface of filamentous bacteriophage was constructed using the SurfZAP vector. The size of the library was approximately 105. The peptide insert was flanked by two cysteines to constrain the peptide structure with a disulfide bond. This library was screened for the topoisomerase II binding peptide. Dramatic enrichment of the fusion phage over the VCS M13 helper phage was demonstrated by bio-panning affinity selection.

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Isolation of Human scFv Antibodies Specific for House Dust Mite Antigens from an Asthma Patient by Using a Phage Display Library

  • Jung, Wang-lim;Lee, Hee-kyung;Yong, Tae-soon;Cha, Sang-hoon
    • IMMUNE NETWORK
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    • v.2 no.2
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    • pp.91-95
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    • 2002
  • Background: In order to characterize human antibodies with specificity for mite allergens at the molecular level, a scFv phage display library was constructed using peripheral blood mononuclear lymphocytes from an asthma patient allergic to mite as Ig gene sources. Methods: Immunoglobulin $V_H$ and V gene fragments were obtained by polymerase chain reaction, and randomly combined in pCANTAB-5E vector. The resulting human scFv phage display library had $3{\times}10^4$ independent clones, and biopanning was performed with house dust mite extracts. Results: Four scFv clones specific for house dust mite extract were isolated. Immunoblot assay showed that our clones reacted to 25 kDa and 50~60 kDa proteins with unknown identity in mite extracts. Sequence analysis indicated that two clones (b7 and c15) are identical, and all clones belong to human $V_H3$ subgroup. On the other hand, light chain usage was different in that two clones (a2 and b7 / c15) belonging to V ${\kappa}4$ subgroup, but a4 used V ${\kappa}1$ light chain gene. Conclusion: Our approach should facilitate provision of useful information on the antibody responses against allergens at the molecular level in humans.

Characterization of Alanine Scanning Mutants of a Peptide Specifically Binding to $TiO_{2}$ Nanoparticles ($TiO_{2}$ Nanoparticle에 특이적으로 결합하는 Peptide의 Alanine Scanning Mutant의 성질에 관한 연구)

  • Seo, Min-Hee;Chael, Hee-Kwon;Myung, Heejoon
    • Microbiology and Biotechnology Letters
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    • v.33 no.4
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    • pp.319-321
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    • 2005
  • We have previously reported the isolation and characterization of peptides binding to $TiO_{2}$ nanoparticles from phage display peptide libraries. One of the peptides (PEP9) was selected and mutant peptide-displaying phages were produced by alanine scanning mutagenesis. The mutant phages were subjected to binding analysis to $TiO_{2}$ nanoparticles. When the proline at residue 4 was substituted by alanine, the binding activity was reduced to $10\%$ of that of wild type PEP9. Substitution of valine at residue 2, serine at residue 3, and isoleucine at residue 5 also decreased the binding to $40\%$. Based on these observations, we concluded that the three dimensional structure generated by residues 2-5 was the critical factor for the binding between PEP9 and the nanoparticle.

Application of Nanoparticles for Materials Recognition using Peptide Phage Display Technique- Part I: Preliminary study using LaPO4 and TiO2 nanoparticles (Peptide phage display 기술을 이용한 나노입자의 materials recognition 응용 - Part I: LaPO4 및 TiO2 나노입자를 이용한 기초연구)

  • Lee, Chang-Woo;Kim, Min-Jung;Standaert, R.;Kim, Seyeon;Owens, E.;Yan, Jun;Choa, Yong-Ho;Doktycz, M.;Lee, Jai-Sung
    • Korean Journal of Metals and Materials
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    • v.46 no.1
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    • pp.6-12
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    • 2008
  • Peptides with specific sequences against $LaPO_4$ and $TiO_2$ nanoparticles were discovered through peptide phage display technique as an application to biomolecular recognition of inorganic materials. Sequencing results showed that a motif consisting of serine and proline was commonly expressed in specific sequences. It was postulated that serine directly bound to nanoparticles using its terminal hydroxyl (OH) group. In this sense, oxygen atom seemed to work as a ligand to metal ions and hydrogen atom as a H-bond donor, was thought to bind to the oxygen atoms or the hydroxyl groups on particle surface. Also, it was expected that proline assists serine to make an ideal van der Waals contact between serine and nanoparticles, which optimizes the binding of peptide onto surface.

Isolation and Characterization of Single-Chain Fv Against Ductal Cells

  • Lee Myung-Hoon;Ryu Hye-Myung;Kim Sun-Zoo;Park Ji-Young;Uhm Ji-Hyun;Park Tae-In
    • Biomedical Science Letters
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    • v.10 no.3
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    • pp.211-217
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    • 2004
  • For discrimination of ductal and ascinar cells, we isolated a single-chain variable domain fragment (scFv) antibody against ductal cells of salivary gland using phage display technique. From the spleen of a mouse immunized with ductal cell lysate, total RNA was prepared and used as a template for cDNA synthesis of antibody genes. The scFv genes were constructed with variable domain genes of heavy and light chain and were introduced into pCANTAB5E to construct phage scFv library. The phage particles specific for acinar cells were screened by subtraction using immunotubes coated with acinar and ductal cell lysate and enzyme-linked immunoabsorbance assay (ELISA). The characteristics of the scFv were determined by immunohistochemistry (IHC) and the result indicated that the isolated scFv has the specificity against ductal cells of salivary glands and tubules of kidney. And the scFv has an unique binding activity specific for Hashimoto's thyroiditis. The nucleotide sequence of isolated scFv gene was determined and revealed that V/sub H/ belongs to the mouse H-chain family subgroup IB and V/sub L/ to the mouse L-chain family subgroup III.

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Expression of the Recombinant Single-Chain Anti-B Cell Lymphoma Antibody

  • Park, Tae-Hyun;Park, Chang-Woon;Awh, Ok-Doo;Lim, Sang-Moo
    • Biomedical Science Letters
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    • v.9 no.3
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    • pp.111-121
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    • 2003
  • Recombinant single chain Fv (scFv) antibodies offer many advantages over mouse monoclonal antibodies such as faster clearance from blood, improved tumor localization, reduced human anti-mouse antibody (HAMA) response, and the availability to manipulate the scFv through genetic approaches. The recombinant phage display was constructed using lym-l hybridoma cells as a source of genetic starting material. mRNA was isolated from the corresponding antibodies hybridoma cells. VH and VL cDNA were amplified with RT-PCR and linked with ScFv by linker DNA to form ScFv DNA, which then were inserted into phagemid pCANTAB5E. The phage of positive clones selected with tube containing raji lymphoma cell and infected by competent E. coli HB2151 to express soluble scFv. The scFv lym-l was secreted into the cytosol and culture supernatant and shown to be of expected size (approximately 32 kDa) by western blot. An active scFv lym-l could be produced in E. coli with soluble form and high yield from hybridoma cell line, using phage display system. Immunoreactivity indicated that scFv lym1 showed a potential biding affinity against the raji lymphoma cell as its parental antibody (intact lym-l Ab).

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Development of a Single Chain Antibody Using a Phage Display Cloning Method for the Detection of 2,4-Dinitrotoluene

  • Na, Jung-Hyun;Joo, Man-Seok;Lee, Won-Kyu;Shim, Hyunbo;Lim, Si-Hyung;Jung, Sang Taek;Yu, Yeon Gyu
    • Bulletin of the Korean Chemical Society
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    • v.34 no.2
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    • pp.460-464
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    • 2013
  • Single-chain variable fragments of antibodies (scFv) specific to 2,4-dinitrotoluene (DNT) were isolated from a phage library displaying synthetic human scFv fragments with 6 diversified complementary determining regions (CDRs). A DNT derivative that contained an extended amine group was synthesized and conjugated to the NHS-group that was linked to magnetic beads. Phages specific to the immobilized DNT derivatives were isolated from the library after 4 rounds of sequential binding and elution processes. The displayed scFv fragments from the isolated phages showed consensus CDR sequences. One DNT-specific scFv was expressed in E. coli and purified using Ni-affinity chromatography. The purified DNT-specific scFv binds specifically to the immobilized DNT-derivative with $K_D$ value of $6.0{\times}10^{-7}$ M. The scFv and DNT interaction was not disrupted by the addition of 4-nitrotoluene or benzoic acid. These data demonstrate that the screened scFv from the phage displayed library could be used for selective and sensitive detection of explosives such as TNT.

Ex12 helper phage improves the quality of a phage-displayed antibody library by ameliorating the adverse effect of clonal variations

  • Choi, Hyo-Jung;Song, Suk-Yoon;Yoon, Jae-Bong;Liu, Li-Kun;Cho, Jae-Youl;Cha, Sang-Hoon
    • BMB Reports
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    • v.44 no.4
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    • pp.244-249
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    • 2011
  • The quality of a phage-displayed antibody library deteriorates with clonal variations, which are caused by differentially expressed Escherichia coli antibody genes. Using the human Fab SP114 against the pyruvate dehydrogenase complex-E2 (PDCE2), we created four E. coli TOP10F' clones with a pCMTG phagemid encoding Fab-pIII (pCMTG-Fab), Fd ($V_H+C_{H1}$)-pIII (pCMTG-Fd), or light chain (L) (pCMTG-L), or the vector only (pCMTG-${\Delta}Fab$) to investigate the effect of clonal variations in a defined manner. Compared to the others, the E. coli clone with pCMTG-Fab was growth retarded in liquid culture, but efficiently produced phage progenies by Ex12 helper phage superinfection. Our results suggest that an antibody library must be cultured for a short duration before helper phage superinfection, and that the Ex12 helper phage helped to alleviate the detrimental effect of clonal variation, at least in part, by preferentially increasing functional phage antibodies during phage amplification.