1999 Fiscal Year Final Research Report Summary
Kinetic screening of phage display libraries by capturing cassette technology.
| Project/Area Number |
09450304
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
生物・生体工学
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| Research Institution | Osaka University |
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Principal Investigator |
FUKUSAKI Eiichiro Graduate School of Engineering, Osaka University, Associate Professor, 大学院・工学研究科, 助教授 (40273594)
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| Co-Investigator(Kenkyū-buntansha) |
KATAKURA Yoshio Graduate School of Engineering, Osaka University, Assistant Professor, 大学院・工学研究科, 助手 (50263207)
YOMO Tetsuya Graduate School of Engineering, Osaka University, Associate Professor, 大学院・工学研究科, 助教授 (00222399)
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| Project Period (FY) |
1997 – 1999
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| Keywords | phage display / kinetics / screening / esterase / nuclease / nucleic acid / saccharide |
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| Research Abstract |
A library of proteins with about 140 amino acid residues containing random sequences was prepared to serve as a model of ancestral proteins, and it was shown that a primordial enzyme may not take a unique conformation but can exert a low catalytic activity. Hence, the known high and specific activity of natural enzymes and the folding ability of natural proteins into unique conformations could have been acquired through the course of evolution. Then, phage libraries displaying artificial proteins with random sequences were constructed for in vitro evolution of the random proteins. Ionic strength was found to be one of the important parameters for screening of phage display libraries. To establish a system in which catalytic proteins are evolutionally selected in vitro, we employed Staphylococcal nuclease (SNase)-DNA as a model of enzyme-substrate. M13 phage displaying active SNase was prepared and modified with N-succinimidy1-6-maleimiddohexanoate. A substrate DNA, of which the both ends are biotinylated and thiolated, respectively, was prepared by PCR. The SNase displaying phage immobilized on a streptavidin beads through the DNA was recovered by activating the displayed SNase with Ca2+. Additionally, we tried to obtain high-affinity DNA ligands, termed "DNA aptamer " using in vitro selection method. A random ssDNA library of 100 bases (59mer random sequence was included) was subjected to the chitin column to obtain the several clones that bound to chitin. Sequencing shown that each clone has the stem loop or the bulge loop structures.
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[Publications] Asao Yamauchi, Tetsuya Yomo, Fumihiro Tanaka, Irfan D. Prijambada, Seiji Ohhashi, Keizo Yamamoto, Yasufumi Shima, Kyoko Ogasahara, Katsuhide Yutani, Mikio Kataoka, Itaru Urabe: "Characterization of soluble artificial with random sequences."FEBS Letters. 421. 147-151 (1998)
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