2002 Fiscal Year Final Research Report Summary
Evolvabtlity of random polypeptides through functional selection within a small library
| Project/Area Number |
12480200
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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 |
Biophysics
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| Research Institution | Osaka University |
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Principal Investigator |
YOMO Tetsuya Department of Bioinformatic Engineering, Graduate School of Information Science and Technology. Associate Professor, 大学院・情報科学研究科, 助教授 (00222399)
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| Project Period (FY) |
2000 – 2002
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| Keywords | random protein / transition state analog / phage display / directed evolution / esterase activity |
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| Research Abstract |
A directed evolution with phage-dispfayed random polypeptides of about 140 amino acid residues was followed until the sixth generation under a selection based on affinity to a transition state analog for an esterase reaction. The experimental design deliberately limits the observation to only 10 clones per generation. The first generation consists of three soluble random polypeptides and seven arbitrarily chosen clones from a previously constructed library. The clone showing the highest affinity in a generation was selected and subjected to random mutagenesis to generate variants for the nest generation. Even within only 10 arbitrarily chosen polypeptides in each of the generations, there are enough variants in accord to capacity of binding affinity. In addition, the binding capacity of the selected polypeptides showed a gradual continuous increase over the generation. Furthermore, the purified selected random polypeptides exhibited a gradual but significant increase in esterase activity. The ease of the functional development within a small sequence variety implies that enzyme evolution is prompted even within a small population of random polypeptides.
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