Construction of a Combinatorial Random Protein Library Displaying on Yeast Cell Surface and Its Application

摘要

A combinatorial protein library was constructed by the method of DNA random-priming, using a novel multicopy cassette vector that can display the heterologous proteins on the cell surface of Saccharomyces cerevisiae by "cell surface engineering". The improved method of DNA random priming was used to generate a large pool of random DNA fragments for increasing the variety of the library. As one of the application of this combinatorial library, an organic solvent-tolerant yeast strain was obtained by screening of this library with n-nonane. Many organic solvents are highly toxic for living organisms because they accumulate in and disrupt cell membranes. Until now, there are few reports on eukaryotic organisms that can adapt to and survive these antimicrobial agents. The acquisition of n-nonane-tolerance was proved to be due to the existence of transformed plasmids. The inserted fragment of this strain was sequenced and the deduced amino acid sequence was evaluated. This strain is the first one to have been endowed artificially with organic solvent-tolerance. The display of combinatorial libraries might open a novel door for molecular breeding of yeasts as well as proteins.