• Journal of Microbiology
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• 제35권4호
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• pp.347-353
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• 1997

We have characterized a phage library displaying random 22mer peptides which were produced as N-terminal fusions to the pIII coat protein of M13 filamentous phages. Among the sixty phages randomly picked from the library, 25 phages had the 22mer peptide inserts. The DNA sequence analysis of the 25 inserts showed the following results: first, each nucleotide was represented almost equally at each codon position except that there were some biases toward G bases at the first position of the codons. Secondly, the expected 47 sense codons were represented. The deduced amino acid sequences of the 25 inserts were analyzed to examine its diversity. Glycine and glutamate were the two most overrepresented residues above the expected value, whereas cysteine and threonine residues were underrepresented. The range of dicersity in dipeptide sequences showed that the amino acid residues were randomly distributed along the peptide insert. Acidic, basic, polar, and nonpolar amino acid residues were represented to the extent expected at most positions of the peptide inserts. The predicted isoelectric points and hydropathy indices of the 25 peptides showed that a variety of the peptide were represented in the library. These results indicate that this phage display library could be useful in fiuding ligands for a broad spectrum of receptors by affinity screening.

• Molecules and Cells
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• 제19권1호
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• pp.54-59
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• 2005

Deoxyribonuclease I (DNase I) is a divalent cation dependent endonuclease and thought to be a significant barrier to effective gene delivery. The only known DNase I-specific inhibitor is monomeric actin which acts by forming a 1:1 complex with DNase I. Its use, however, is restricted because of tendency to polymerize under certain conditions. We screened two random phage peptide libraries of complexity $10^8$ and $10^9$ for DNase I binders as candidates for DNase I inhibitors. A number of DNase I-binding peptide sequences were identified. When these peptides were expressed as fusion proteins with Escherichia coli maltose binding protein, they inhibited the actin-DNase I interaction ($IC_{50}=0.1-0.7{\mu}M$) and DNA degradation by DNase I ($IC_{50}=0.8-8{\mu}M$). Plasmid protection activity in the presence of DNase I was also observed with the fusion proteins. These peptides have the potential to be a useful adjuvant for gene therapy using naked DNA.

• IMMUNE NETWORK
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• 제2권2호
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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.

• Journal of Microbiology and Biotechnology
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• 제21권2호
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• pp.197-203
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• 2011

Lactobacilli are normal constituents of the intestinal microbiota, and some strains show the capacity to bind to extracellular matrix proteins and components of the mucosal layer, which represents an adaptation to persist in this niche. A shotgun phage-display library of Lactobacillus casei BL23 was constructed and screened for peptides able to bind to fibronectin and collagen. Clones showing binding to these proteins were isolated, which encoded overlapping fragments of a putative transcriptional regulator (LCABL_29260), a hypothetical protein exclusively found in the L. casei/rhamnosus group (LCABL_01820), and a putative phage-related endolysin (LCABL_13470). The construction of different glutathione S-transferase (GST) fusions confirmed the binding activity and demonstrated that the three identified proteins could interact with fibronectin, fibrinogen, and collagen. The results illustrate the utility of phage display for the isolation of putative adhesins in lactobacilli. However, it remains to be determined whether the primary function of these proteins actually is adhesion to mucosal surfaces.

• 한국식품저장유통학회지
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• 제15권5호
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• pp.617-621
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• 2008

Sequential passes through $Sephadex^{TM}$ columns were used to select phages that displays ligands for dextran ($\alpha$-1,6 linked linear chains) from a phage antibody library. Those phages that bound to the $Sephadex^{TM}$ in each iteration were replicated in E. coli. A phage preparation isolated on the third round selection produced 5.4 nephelos turbidity units (NTU) in a dextran specific immunonephelometric assay, a 2.2 fold higher value than the phage preparation from the first round selection. This phage gave $72\;{\pm}\;10$ normalized intensity (N.I.) in a dip-stick assay against high molecular size dextran (T2000, $2\;{\times}\;10^6) and significantly lower color ($30\;{\pm}\;6$ N.I.) against low molecular size dextran (T10, $10^4$). The presence of an Fab insert in each of these phages was confirmed using a $\beta-galactosidase linked assay and polymerase chain reaction.

• Bulletin of the Korean Chemical Society
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• 제31권12호
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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.

• 한국미생물·생명공학회지
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• 제35권4호
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• pp.278-284
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• 2007

생체 염 농도에서도 항균활성을 유지할 수 있는 선형 ${\alpha}$-helical 항균 펩타이드를 M13 펩타이드 라이브러리로부터 탐색할 수 있는 새로운 방법을 개발하였다. M13의 pIII은 magainin 유도체인 MSI-344와 indolicidin과 융합된 상태에서도 파아지의 viability에 영향을 주지 않는 것으로 보아, MSI-344와 indolicidin의 대장균에 대한 독성을 중화할 수 있는 것으로 판단되며, 따라서 대장균에서 항균 펩타이드 라이브러리의 제조가 가능함을 증명하였다. 선형 항균 펩타이드의 보존된 부위를 바탕으로, 13개의 아미노산 잔기로 구성된 semi-combinatorial 항균 펩타이드 라이브러리를 M13를 이용하여 제조하였다. 제조된 파아지 라이브러리는 먼저 적혈구에 흡착시켜, 높은 용혈 역가를 가질 가능성이 있는 파아지를 제거한 후, 높은 염 농도에서 Pseudomonas aeruginosa와 Staphylococcus aureus에 흡착할 수 있는 파아지를 탐색하였다. 탐색된 펩타이드들은 염이 없는 조건에서는 비교적 낮은 항균 역가를 보였지만, P06와 S18 펩타이드의 경우, 생체 염 농도보다 높은 150 mM $Na^+$, 2 mM $Mg^{2+}$, 2 mM $Ca^{2+}$의 조건에서도 항균 역가가 영향을 받지 않았으며, 심각한 용혈 역가 또한 보이지 않았다. 본 연구에서 개발한 대상 세균에 대한 흡착능력을 이용한 탐색방법은 salt-tolerant antimicrobial peptide의 개발의 새로운 가능성을 제시하였다.

• IMMUNE NETWORK
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• 제1권1호
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• pp.77-86
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• 2001

Background: Hepatitis C virus(HCV), a family of Flaviviridae, has a host cell-derived envelope containing a positive-stranded RNA genome, and has been known as the maj or etiological agent for chronic hepatitis, hepatic cirrhosis, and hepatocellular carcinoma. There remains a need to dissect a molecular mechanism of pathogenesis for the development of therapeutic and effective preventive measure for HCV. Identification of cellular receptor is of central importance not only to understand the viral pathogenesis, but also to exploit strategies for prevention of HCV. This study was aimed at identifying peptide mimotopes inhibiting the binding of E2 protein of HCV to MOLT-4 cell. Methods: In this study, phage peptide library displaying a random peptides consisting of 7 or 12 random peptides was employed in order to pan against E2 protein. Free HCV particles were separated from the immune complex forms by immunoprecipitation using anti-human IgG antibody, and used for HCV-capture ELISA. To identify the peptides inhibiting E2-binding to MOLT-4 cells, E2 protein was subj ect to bind to MOLT-4 cells under the competition with phage peptides. Results: Several phage peptides were selected for their specific binding to E2 protein, which showed the conserved sequence of SHFWRAP from 3 different peptide sequences. They were also able to recognize the HCV particles in the sera of HCV patients captured by monoclonal antibody against E2 protein. Two of them, showing peptide sequence of HLGPWMSHWFQR and WAPPLERSSLFY respectively, were revealed to inhibit the binding of E2 protein to MOLT-4 cell efficiently in dose dependent mode. However, few membrane-associated receptor candidates were seen using Fasta3 programe for homology search with these peptides. Conclusion: Phage peptides containing HLGPWMSHWFQR and WAPPLERSSLFY respectively, showed the inhibition of E2-binding to MOLT-4 cells. However, they did not reveal any homologues to cellular receptors from GenBank database. In further study, cellular receptor could be identified through the screening of cDNA library from MOLT-4 or hepatocytes using antibodies against these peptide mimotopes.

• Molecules and Cells
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• 제27권2호
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• pp.225-235
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• 2009

Antibody phage display provides a powerful and efficient tool for the discovery and development of monoclonal antibodies for therapeutic and other applications. Antibody clones from synthetic libraries with optimized design features have several distinct advantages that include high stability, high levels of expression, and ease of downstream optimization and engineering. In this study, a fully synthetic human scFv library with six diversified CDRs was constructed by polymerase chain reaction assembly of overlapping oligonucleotides. In order to maximize the functional diversity of the library, a ${\beta}$-lactamase selection strategy was employed in which the assembled scFv gene repertoire was fused to the 5'-end of the ${\beta}$-lactamase gene, and in-frame scFv clones were enriched by carbenicillin selection. A final library with an estimated total diversity of $7.6{\times}10^9$, greater than 70% functional diversity, and diversification of all six CDRs was obtained after insertion of fully randomized CDR-H3 sequences into this proofread repertoire. The performance of the library was validated using a number of target antigens, against which multiple unique scFv sequences with dissociation constants in the nanomolar range were isolated.

• KSBB Journal
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• 제28권3호
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• pp.185-190
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• 2013

For the selection of peptide specifically binding to $Pb^{2+}$, the biopanning with the commercially available Ph.D.-7 phage displayed heptapeptide library was carried out against $Pb^{2+}$ immobilized on a metal-chelating IDA (iminodiacetic acid) resin. After four rounds of screening against $Pb^{2+}$-IDA including negative selections against charged bead with metal ions other than $Pb^{2+}$ and uncharged bead, several candidate lead-binding phage peptides were initially determined based on the order of frequency from the screened phage clones. Of the selected phage peptide sequences, the peptide of the highest frequency, CysSerIleArgThrLeuHisGlnCys (CSIRTLHQC) also exhibited the strongest affinity for $Pb^{2+}$ in binding assays for individual phage clones. However, there was not a significant difference in $Pb^{2+}$ affinity between selected peptides when using synthetic heptapeptides corresponding to the displayed peptide sequences of phage clones.