Development of production method of large size of combinatorial libraries by mating of Sccharomyces cerevisiae cells and screening

近年来，各种生物活性材料被广泛应用于许多生物领域。为了确保通过筛选获得具有所需特性的生物活性材料，有必要对这些生物材料建立庞大的文库。然而，文库中的物种数量是有限的，这取决于文库的构建方法。在本工作中，我们试图设计一种新的方法，利用酵母细胞的交配特性来生产更大规模的组合文库。酿酒酵母中有两种交配型，即a型和α型，它们可以形成二倍体，同时携带来自a型和α型的基因。例如，通过将重链和轻链的基因库分别转换为a-型和α-型，应该可以通过交配获得更大规模的抗体库。此外，由于产生的抗体可以被设计为从转化子中分泌，因此可以很容易地筛选出产生抗体的菌株。为了验证我们的方法，将含有人抗外毒素重链基因(μ)和轻链基因(κ)的表达载体分别转化到酿酒酵母A型和α型菌株，并将这两种类型的培养细胞混合进行交配。证实了完全组装的抗体的分泌。结果表明，该方法可以使图书馆规模扩大一倍。此外，还研究了培养基、细胞浓度和A型细胞与α细胞的比例对交配效率的影响。

Recent years a variety of bioactive materials are widely used in many biological fields. To make sure obtaining bioactive materials with desired characteristics by screening, it is necessary to make huge libraries of these biomaterials. Numbers of species in a library, however, are limited depending on construction methods of libraries.In this work we try to devise a new method for production of a larger size of combinatorial libraries by use of mating characteristics of yeast cells. There are two kinds of mating types in S. cerevisiae, a-type and α-type, and they can make a diploid, which carries both the genes originated from a-type and α-type. For example, by transforming libraries of genes of heavy and light chains to a- and α-types respectively, it should be possible to obtain a larger size of libraries of antibodies by mating them. Further, since antibodies produced can be designed to be secreted from transformants, straines for production of antibodies may be easily screened.To confirm our method, the expression vectors containing heavy chain gene (μ) and light chain genes (κ) of human antibody against exotoxin were transformed into a-type and α-type strains of S. cerevisiae, respectively and these transformants were mated by mixing the cultured cells of both types. Secretion of completely assembled antibody was confirmed. It was shown that a library can be doubled by this method. Further, the effects of media, cell concentration and the ratio of a-type cell to α-type cell on the mating efficiency were studied.