Construction of combinatorial protein library and ultra-speedy microscreening

利用酵母细胞进行细胞表面工程已经在生物技术的许多领域得到了发展。与噬菌体展示系统相比，酵母细胞表面可以展示更大分子尺寸的活性肽和蛋白质，但噬菌体展示系统的转化效率更高。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一个新领域。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对蛋白质结构-功能关系的组合分析，并构建实用的蛋白质工程系统。此外，从随机DNA序列中创造完全新颖和功能性蛋白质的可能性已经得到证明。随着分子展示系统和高通量系统的发展，对多种基因和人工合成DNA衍生的蛋白质功能的分析越来越多，蛋白质组分析和蛋白质文库构建的方法也得到了发展。这些系统的结合有望使DNA数据和蛋白质功能的简单和同步分析成为可能，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，这集中在蛋白质工程的创新和新蛋白质的创造上。为了进一步加深对蛋白质功能的了解，进一步的方法创新是必要的。基于“组合生物工程”中描述的分子显示系统，改变了以往的蛋白质工程方法。该方法将蛋白质工程研究策略从单个点的突变提高到结构信息组合中的多个点和组合点的突变。该方法首先构建目标结构域和区域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，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，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。此外，不需要单独纯化突变蛋白。具有突变蛋白的全细胞生物催化剂在培养后很容易制备。这些创新的方法有望在未来引领蛋白质工程的突破。少

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