2004 Fiscal Year Final Research Report Summary
Construction of combinatorial protein library and ultra-speedy microscreening
| Project/Area Number |
15380230
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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 |
Applied molecular and cellular biology
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| Research Institution | KYOTO UNIVERSITY |
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Principal Investigator |
UEDA Mitsuyoshi Kyoto University, Graduate School of Agriculture, 農学研究科, 教授 (90183201)
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| Project Period (FY) |
2003 – 2004
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| Keywords | Cell surface engineering / Cell surface display / Microchamber array / Protein library / Nanotechnology / Combinatorial mutagenesis / Single cell PCR method / Microscreening |
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| Research Abstract |
Cell-surface engineering using yeast cells has developed in many fields of biotechnology. Active peptides and proteins with larger size of molecules can be displayed on the yeast-cell surface than with the phage display system, although the latter has greater transformation efficiency. Because of these aspects, the yeast-cell surface engineering system represents a novel field of protein engineering and protein creation. As the yeast display system allows active enzymes with various sizes and forms to be displayed, it is expected that a combination with crystallization analysis and computerized modeling will facilitate combinatorial analysis of the structure-function relationship of proteins and the construction of a practical protein-engineering system. Furthermore, the possibility of creating completely novel and functional proteins from random DNA alignments has been demonstrated. In conjunction with molecular display systems and high-throughput systems for combinatorial and speedy
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analysis of the functions of proteins derived from many genes and artificially synthesized DNA, methods of proteome analysis and protein-library construction have been also developed. The combination of these systems is expected to make possible easy and simultaneous analysis of DNA data and protein function and to greatly support the combination of genomics with proteomics. This concentrates, among these developments, on innovation in protein engineering and on the creation of novel proteins. To further advance understanding of protein functions, further innovation in methodology has become necessary. Based on the molecular display system described in "Combinatorial Bioengineering", previous methods of protein engineering have changed. The novel method has led to the improvement of protein-engineering research strategies from mutagenesis of individual points to mutagenesis of multiple and combinatorial points in the combination of structural information. This method begins with the construction of a protein library with continuous or non-continuous combinatorial mutation of target domains and regions. Next, direct screening of target clones with a high-throughput system becomes possible. In the case of the yeast display system, the correspondence between the genotype (introducing the gene) and the phenotype (expressing the gene) becomes clear by the determination of the DNA sequence encoding the displayed proteins by simply providing primers on either side of the introduced gene. Furthermore, it is not necessary to purify the mutated proteins individually. Whole-cell biocatalysts with mutated proteins can thus be prepared easily after cultivation. These innovative methods are expected to lead breakthroughs in protein engineering in the future. Less
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