Construction of combinatorial protein library and ultra-speedy microscreening

组合蛋白库的构建和超快速微筛选

• 批准号: 15380230
• 负责人: UEDA Mitsuyoshi
• 金额: $ 10.3万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15380230/
• 关键词: Cell surface engineering; Cell surface display; Microchamber array; Protein library; Nanotechnology; Combinatorial mutagenesis; Single cell PCR method; Microscreening; ナノテクノロジ; 細胞表層デイスプレイ; タンパク質ライラリー

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 … More 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

使用酵母细胞的细胞表面工程已经在许多生物技术领域发展。具有更大尺寸分子的活性宠物和蛋白质可以在酵母细胞表面显示，而不是噬菌体显示系统，尽管后者具有更大的转化效率。由于这些方面，酵母菌表面工程系统代表了蛋白质工程和蛋白质创造的新领域。由于酵母显示系统允许显示各种大小和形式的活性酶，因此预计结合结晶分析和计算机建模的组合将有助于蛋白质结构 - 功能关系的组合分析以及实用蛋白质引擎系统的结构。此外，已经证明了从随机DNA比对创建完全新颖和功能性蛋白质的可能性。结合了用于组合和快速组合的分子显示系统和高通量系统……对源自许多基因和人为合成的DNA的蛋白质功能进行了更多分析，还开发了蛋白质分析和蛋白质外生结构的方法。这些系统的组合有望使对DNA数据和蛋白质功能的简单简单分析，并在很大程度上支持基因组学与蛋白质的组合。在这些发展中，这种浓度是关于蛋白质工程和新蛋白质创造的创新。为了进一步提高人们对蛋白质功能的了解，方法论已经有必要进一步创新。基于“组合生物工程”中描述的分子显示系统，蛋白质工程的先前方法已经改变。这种新方法导致蛋白质工程研究策略从单个点的诱变到结构信息组合的多个和组合点的诱变。该方法始于构建目标域和区域的蛋白质文库，该蛋白质文库具有连续或不连续的组合突变。接下来，将可以直接筛选具有高通量系统的目标克隆。在酵母显示系统的情况下，基因型（引入基因）与表型（表达基因）之间的对应关系通过确定编码所带来的蛋白质的DNA序列通过简单地在引入基因的任一侧提供引物来清除。此外，不必单独净化突变的蛋白质。因此，具有突变蛋白的全细胞生物催化剂可以在培养后很容易制备。这些创新的方法有望在将来引起蛋白质工程的突破。较少的

项目成果

Display of functional hetero-plogomeric catalytic antibody on the yeast cell surface

在酵母细胞表面展示功能性异源催化抗体

• 发表时间: 2003
• 期刊: Appl.Mocrobiol.Biotechnol. 62
• 作者: K.Inanaga;K.Takasu;M.Ihara;Y.Lin et al.
• 通讯作者: Y.Lin et al.

S.Shibasaki et al.: "Development of combinatorial bioengineering using yeast cell"Biosensors & Bioelectronics. 19・2. 123-130 (2003)

S. Shibasaki 等：“使用酵母细胞的组合生物工程的开发”《生物传感器与生物电子学》19・2（2003）。

Comparison of two forms of catalytic antibody displayed on yeast

酵母上展示的两种形式的催化抗体的比较

• 发表时间: 2004
• 期刊: J.Mol.Catalys. 28・4-6
• 作者: Kuwabara M.;Takahashi K;Inanami O.;Y.Lin et al.
• 通讯作者: Y.Lin et al.

Y.Lin et al.: "Display of a functional hetero-oligomeric catalytic antibody"Appl.Microbiol.Biotechnol.. 62・2-3. 226-232 (2003)

Y.Lin等：“功能性异源寡聚催化抗体的展示”Appl.Microbiol.Biotechnol.. 62·2-32(2003)。

植田充美: "抗体酵素の新しいディスプレイ法の開拓"BIO INDUSTRY. 20・7. 15-22 (2003)

上田光美：“抗体酶的新展示方法的开发”BIO INDUSTRY 20・7（2003）。