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生物技术的许多领域中发展。与噬菌体展示系统相比，具有更大分子尺寸的活性肽和蛋白质可以展示在酵母细胞表面上，尽管后者具有更高的转化效率。由于这些方面，酵母细胞表面工程系统代表了蛋白质工程和蛋白质创造的新领域。由于酵母展示系统可以展示各种大小和形式的活性酶，因此，结合结晶分析和计算机建模将有助于蛋白质结构-功能关系的组合分析和实用蛋白质工程系统的构建。此外，已经证明了从随机DNA比对中产生完全新颖的功能性蛋白质的可能性。结合分子展示系统和高通量系统， ...更多信息 在对来自许多基因和人工合成DNA的蛋白质的功能进行分析的基础上，还开发了蛋白质组分析和蛋白质文库构建的方法。这些系统的组合有望使DNA数据和蛋白质功能的简单和同时分析成为可能，并极大地支持基因组学与蛋白质组学的结合。在这些发展中，这集中在蛋白质工程的创新和新蛋白质的创造上。为了进一步推进对蛋白质功能的理解，有必要在方法学上进行进一步的创新。基于“组合生物工程”中描述的分子展示系统，以前的蛋白质工程方法发生了变化。这种新的方法导致了蛋白质工程研究策略的改进，从单个点的诱变到结构信息组合中的多个和组合点的诱变。该方法首先构建具有目标结构域和区域连续或非连续组合突变的蛋白质库。接下来，用高通量系统直接筛选靶克隆成为可能。在酵母展示系统的情况下，基因型（引入基因）和表型（表达基因）之间的对应性通过简单地在引入基因的任一侧提供引物来确定编码展示蛋白质的DNA序列而变得清楚。此外，不需要单独纯化突变蛋白。因此，具有突变蛋白质的全细胞生物催化剂可以在培养后容易地制备。这些创新方法有望在未来引领蛋白质工程的突破。少

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）。

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)。

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.

Isolation of novel catalytic antibody clones from library displayed on yeast

从酵母展示的文库中分离新型催化抗体克隆

• 发表时间: 2004
• 期刊: J.Mol.Catalys. 28・4-6
• 作者: Imoto S.;Haruta Y.;Watanabe K.;Sasaki S.;Y.Lin et al.
• 通讯作者: Y.Lin et al.