• 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.

• Journal of Microbiology
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• v.42 no.3
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• pp.228-232
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• 2004

In this paper, we investigated the development of a simplified and rapid primary capture step for the recovery of M13 bacteriophage from particulate-containing feedstock. M13 bacteriophage, carrying an insert, was propagated and subsequently purified by the application of both conventional multiple steps and expanded bed anion exchange chromatography. In the conventional method, precipitation was conducted with PEG/NaCl, and centrifugation was also performed. In the single step expanded bed anion exchange adsorption, UpFront FastLine$\_$TM/20 (20mm i.d.) from UpFront Chromatography was used as the contactor, while 54$m\ell$ (H$\_$o/=15cm) of STREAMLINE DEAE (p=1.2 g/㎤) from Amersham Pharmacia Biotechnology was used as the anion exchanger. The performance of the two methods were evaluated, analysed, and compared. It was demonstrated that the purification of the M13 bacteriophage, using expanded bed anion exchange adsorption, yielded the higher recovery percentage, at 82.86％. The conventional multiple step method yielded the lower recovery percentage, 36.07％. The generic application of this integrated technique has also been assessed.

• Interdisciplinary Bio Central
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• v.4 no.3
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• pp.9.1-9.7
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• 2012

Filamentous phages have been in the limelight as a new type of nanomaterial. In this study, genetically and chemically modified fd phage was used to generate a biomimetic phage self-assembly product. Positively charged fd phage (p8-SSG) was engineered by conjugating 3-aminopropyltriethoxysilane (APTES) to hydroxyl groups of two serine amino acid residues introduced at the N-terminus of major coat protein, p8. In particular, formation of a phage network was controlled by changing mixed ratios between wild type fd phage and APTES conjugated fd-SSG phage. Assembled phages showed unique bundle and network like structures. The bacteriophage based self-assembly approach illustrated in this study might contribute to the design of three dimensional microporous structures. In this work, we demonstrated that the positively charged APTES conjugated fd-SSG phages can assemble into microstructures when they are exposed to negatively charged wild-type fd phages through electrostatic interaction. In summary, since we can control the phage self-assembly process in order to obtain bundle or network like structures and since they can be functionalized by means of chemical or genetic modifications, bacteriophages are good candidates for use as bio-compatible scaffolds. Such new type of phage-based artificial 3D architectures can be applied in tuning of cellular structures and functions for tissue engineering studies.

• 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.