Construction of combinatorial protein library and ultra-speedy microscreening

使用酵母细胞的细胞表面工程已在生物技术的许多领域得到发展。与噬菌体展示系统相比，分子尺寸更大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尺寸和形式的活性酶，因此结晶分析和计算机建模的结合有望促进蛋白质结构与功能关系的组合分析以及实用蛋白质工程系统的构建。此外，通过随机 DNA 比对创造全新的功能性蛋白质的可能性已经得到证明。结合分子展示系统和高通量系统，对来自许多基因和人工合成 DNA 的蛋白质功能进行组合和快速分析，还开发了蛋白质组分析和蛋白质文库构建方法。这些系统的结合有望使 DNA 数据和蛋白质功能的轻松同步分析成为可能，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，重点是蛋白质工程的创新和新型蛋白质的创造。为了进一步加深对蛋白质功能的理解，有必要在方法论上进一步创新。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改变。该新方法导致蛋白质工程研究策略从单个点诱变到结构信息组合中的多个和组合点诱变的改进。该方法首先构建具有目标结构域和区域的连续或非连续组合突变的蛋白质文库。接下来，用高通量系统直接筛选目标克隆成为可能。在酵母展示系统的情况下，通过简单地在引入基因的两侧提供引物来确定编码展示蛋白质的DNA序列，基因型（引入基因）和表型（表达基因）之间的对应关系变得清晰。此外，没有必要单独纯化突变蛋白。因此，在培养后可以轻松制备具有突变蛋白的全细胞生物催化剂。这些创新方法有望在未来引领蛋白质工程的突破。较少的

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