• 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.

• YAKHAK HOEJI
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• v.47 no.3
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• pp.176-179
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• 2003

A library of unlimited number of novel lectins with diverse specificities has been previously generated by randomly mutating the carbohydrate-recognition domain of Maackia amurensis hemagglutinin (MAH). To establish the experimental environment capable of selecting high affinity mutant lectins in E. coli, phage display system was adapted. Carbohydrate binding capacity of two phagemid vectors, pComb3 and pComb8 displaying wild-type MAH lectin was assessed. Specific bindings of pComb3 and pComb8 phages expressing w.t. MAH to affinity-purified polyclonal anti-MAH antibody and to glycophorin was demonstrated. Both phages also showed strong hemagglutinating activity to intact but not sialidase-treated human erythrocytes, which is consistent to the specificity of native MAH. Taken together, two different phage display vectors successfully allowed the expression of active MAH as a fusion protein on the surface of bacteriophage, which will lead to preparation of unique plant lectins with high affinity toward a variety of carbohydrate chains.

• Journal of Microbiology
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• v.35 no.4
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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.

• BMB Reports
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• v.33 no.3
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• pp.242-248
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• 2000

Phage display of proteins is a powerful tool for protein engineering since a vast library of sequences can be rapidly screened for a specific property. In this study, we develop da new phage display vector that was derived from a pET-25b(+) vector. The pET-25b(+) was modified in order that the expressed protein would have a T7-tag at the amino terminus and GpS (a major coat protein of M13 phage) at the carboxyl terminus. Another vector without the gp8 gene was also constructed. The newly developed phagemid vectors have several advantageous features. First, it is easy to examine whether or not the target proteins are functional and faithfully transported into the periplasmic space. This feature is due to the fact that recombinant proteins are produced abundantly in the pET system. Second, the T7-tag makes it possible to detect any target proteins that are displayed on the surface of filamentous bacteriophage. To verify the utility of the vector, the clones containing the glutathione S-transferase (GST) gene as a target were examined. The result showed that the GST produced from the recombinant vector was successfully transported into the periplasmic space and had the anticipated enzyme activity. Western blot analysis using a T7-tag antibody also showed the presence of the target protein displayed on the surface of the phage. The phages prepared from the recombinant clones were able to bind to glutathione-Sepharose and then eluted with glutathione. These results showed that the new vectors developed in this study are useful for the phage display of proteins.

• BMB Reports
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• v.38 no.3
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• pp.290-293
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• 2005

In order to investigate the epitope of basic fibroblast growth factor (bFGF) and its immunogenicity, the epitopes of bFGF were screened from the phage display library with monoclonal antibody GF22, which can neutralize the bio-activity of bFGF. By three rounds of screening, the positive phage clones with bFGF epitopes were selected, which can effectively block the bFGF to bind with GF22. Sequence analysis showed that the epitopes shared a highly conservative sequence (Leu-Pro-Pro/Leu-Gly-His-Phe/Ile-Lys). The sequence of PPGHFK was located at 22-27 of the bFGF. The specific immuno-response of mouse could be highly induced by phage clones with the epitopes. And the anti-bFGF activity induced by LPGHFK was 3 times higher than the original sequence, which showed that the mimetic peptide LPLGHIK might be used as a tumor vaccine in the prevention and treatment of tumor.

• Molecules and Cells
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• v.27 no.2
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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.

• Microbiology and Biotechnology Letters
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• v.35 no.4
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• pp.278-284
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• 2007

A novel screening strategy for salt-resistant antimicrobial peptides from a M13 peptide library was developed. Fusion of MSI-344, a magainin derivative and indolicidin to pIII coat proteins did not significantly affect viability of the recombinant phages, which indicated that the pIII could neutralize toxicity of the antimicrobial peptides and therefore it is possible to construct antimicrobial peptide library in Escherichia coli. On the basis of the conserved sequence of ${\alpha}$-helical antimicrobial peptides, a semi-combinatorial peptide library was constructed in which the peptides were displayed by pIII. To remove hemolytic activity from the library, the phages bound to red blood cells were removed, and the subtracted phage library was screened for binding to target bacteria Pseudomonas aeruginosa and Staphylococcus aureus under high salt concentrations. The screened peptides showed relatively low antimicrobial activity against the target bacteria. However, antimicrobial activities of the screened peptides P06 and S18 were not affected by the cation concentrations of 150 mM $Na^+$, 2 mM $Mg^{2+}$ and 2 mM $Ca^{2+}$ without significant hemolytic activity. This screening strategy that is based on binding capacity to target cells provides new potential to develop salt-tolerant antimicrobial peptides.

• Journal of Microbiology
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• v.45 no.6
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• pp.528-533
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• 2007

In a previous study we generated an anti-Hepatitis B Virus (HBV) preS1 humanized antibody (HzKR127) that showed in vivo HBV-neutralizing activity in chimpanzees. However, the antigen-binding affinity of the humanized antibody may not be sufficient for clinical use and thus affinity maturation is required for better therapeutic efficacy. In this study, phage display technique was employed to increase the affinity of HzKR127. All six amino acid residues (Glu95-Tyr96-Asp97-Glu98-Ala99-Tyr100) in the heavy (H) chain complementary-determining region 3 (HCDR3) of HzKR127 were randomized and phage-displayed single chain Fv (scFv) library was constructed. After three rounds of panning, 12 different clones exhibiting higher antigen-binding activity than the wild type ScFv were selected and their antigen-binding specificity for the preS1 confirmed. Subsequently, five ScFv clones were converted to whole IgG and subjected to affinity determination. The results showed that two clones (B3 and A19) exhibited an approximately 6 fold higher affinities than that of HzKR127. The affinity-matured humanized antibodies may be useful in anti-HBV immunotherapy.

• BMB Reports
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• v.37 no.2
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• pp.159-166
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• 2004

Angiostatin is a potent anti-angiogenic protein. To examine the angiostatin-interacting proteins, we used the display-cloning method with a T7 phage library presenting human cDNAs. The specific T7 phage clone that bound to the immobilized angiostatin was isolated, and a novel gene encoding the displayed polypeptide on the isolated T7 phage was identified. The displayed angiostatin-binding sequence was expressed in E. coli as a soluble protein and purified to homogeneity. This novel angiostatin-binding region interacted specifically to angiostatin with a dissociation constant of $3.4{\times}10^{-7}\;M$. A sequence analysis showed that the identified sequence was a part of the large ORF of 1,998 amino acids, whose function has not yet been characterized. A Northern analysis indicated that the gene containing the angiostatin-binding sequence was expressed differentially in the developmental stages or cell types.

• The Korean Journal of Physiology and Pharmacology
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• v.28 no.5
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• pp.457-467
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• 2024

Nanobodies derived from camelids and sharks offer unique advantages in therapeutic applications due to their ability to bind to epitopes that were previously inaccessible. Traditional methods of nanobody development face challenges such as ethical concerns and antigen toxicity. Our study presents a synthetic, phage-displayed nanobody library using trinucleotide-directed mutagenesis technology, which allows precise amino acid composition in complementarity-determining regions (CDRs), with a focus on CDR3 diversity. This approach avoids common problems such as frameshift mutations and stop codon insertions associated with other synthetic antibody library construction methods. By analyzing FDA-approved nanobodies and Protein Data Bank sequences, we designed sub-libraries with different CDR3 lengths and introduced amino acid substitutions to improve solubility. The validation of our library through the successful isolation of nanobodies against targets such as PD-1, ATXN1 and STAT3 demonstrates a versatile and ethical platform for the development of high specificity and affinity nanobodies and represents a significant advance in biotechnology.