• Asian Pacific Journal of Cancer Prevention
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• 제13권1호
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• pp.377-381
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• 2012

The aim of this study was to screen for polypeptides binding specifically to LoVo human colorectal cancer cells using a phage-displayed peptide library as a targeting vector for colorectal cancer therapy. Human normal colorectal mucous epithelial cells were applied as absorber cells for subtraction biopanning with a c7c phage display peptide library. Positive phage clones were identified by enzyme-linked immunosorbent assay and immunofluorescence detection; amino acid sequences were deduced by DNA sequencing. After 3 rounds of screening, 5 of 20 phage clones screened positive, showing specific binding to LoVo cells and a conserved RPM motif. Specific peptides against colorectal cancer cells could be obtained from a phage display peptide library and may be used as potential vectors for targeting therapy for colorectal cancer.

• 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.

• BMB Reports
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• 제33권3호
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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.

• Journal of Microbiology
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• 제37권2호
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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.

• BMB Reports
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• 제39권1호
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• pp.55-60
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• 2006

We describe a phage display strategy, based on the differential resistance of proteins to denaturant-induced unfolding, that can be used to select protein variants with improved conformational stability. To test the efficiency of this strategy, wild-type and two stable variants of ${\alpha}_1$-antitrypsin (${\alpha}_1AT$) were fused to the gene III protein of M13 phage. These phages were incubated in unfolding solution containing denaturant (urea or guanidinium chloride), and then subjected to an unfavorable refolding procedure (dialysis at $37^{\circ}C$). Once the ${\alpha}_1AT$ moiety of the fusion protein had unfolded in the unfolding solution, in which the denaturant concentration was higher than the unfolding transition midpoint ($C_m$) of the ${\alpha}_1AT$ variant, around 20% of the phage retained binding affinity to anti-${\alpha}_1AT$ antibody due to a low refolding efficiency. Moreover, this affinity reduced to less than 5% when 10 mg/mL skimmed milk (a misfolding-promoting additive) was included during the unfolding/refolding procedure. In contrast, most binding affinity (>95%) remained if the ${\alpha}_1AT$ variant was stable enough to resist unfolding. Because this selection procedure does not affect the infectivity of M13, the method is expected to be generally applicable to the high-throughput screening of stable protein variants, when activity-based screening is not possible.

• 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.

• Journal of Microbiology and Biotechnology
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• 제16권11호
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• pp.1784-1790
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• 2006

Bacillus anthracis is a causative agent of anthrax. Anthrax toxins are composed of a protective antigen (PA), lethal factor (LF), and edema factor (EF), in which the PA is a central mediator for the delivery of the two enzymatic moieties LF and EF. Therefore, the PA has been an attractive target in the prevention and vaccinization for anthrax toxin. Recently, it has been reported that the molecule consisting of multiple copies of PA-binding peptide, covalently linked to a flexible polymer backbone, blocked intoxification of anthrax toxin in an animal model. In the present study, we have screened novel diverse peptides that bind to PA with a high affinity (picomolar range) from an M13 peptide display library and characterized the binding regions of the peptides. Our works provide a basis to develop novel potent inhibitors or diagnostic probes with a diverse polyvalence.

• 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.

• BMB Reports
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• 제44권4호
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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.

• 약학회지
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• 제57권2호
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• pp.125-131
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• 2013

Biologically active peptides, including growth factors and cytokines, participate in various biological processes in human skin. They could provide a great advantage of maintaining healthy skin. Many peptide growth factors like epidermal growth factor (EGF) and human growth hormone (hGH) have been used in cosmetic formulations. The delivery of peptide growth factors across the Stratum corneum, however, seems not sufficient because of their physical properties such as high molecular weight and hydrophilicity. So increasing the penetration of growth factors of interest into skin would be a major concern for ensuring their maximum biological efficacy. In this study, we have identified several skin penetration-enhancing peptides which facilitate delivery of growth factors, when fused at N-terminus of the target protein, into skin. For efficient and rapid screening, we constructed a skin-penetrating assay system using Franz cell and porcine skin. Next, we carried out phage display screening using M-13 bacteriophage with random 12 -amino acid library on its coat protein P3 on that system. After several selection rounds, peptide sequences facilitate the penetration of phages through the porcine skin were identified from a large population of phages. We found that phages with the most potent peptide (S3-2, NGSLNTHLAPIL) could penetrate the porcine skin eight times more than those with control peptide (12 mino acids scrambled peptide). Furthermore, growth factors conjugated with S3-2 peptide penetrate porcine skin three to five times efficiently than non-conjugated growth factors. In conclusion, our data shows that the skin penetration-enhancing peptide we have characterized could increase the delivery of growth factors and is useful for cosmeceutical application.