Abstract
BACKGROUND: The method of protein-protein interaction analysis using the yeast two-hybrid system in Saccharomyces cerevisiae cells is used to search for protein coregulators. This method is also used for mass screening of libraries of cloned fragments of complementary DNA (cDNA) that are translated in the cell. The key factor in the success of such screening is the level of efficiency of yeast cell transformation, since the resolution of the analysis is based on this. AIM: The aim of this study is to search for optimal parameters for chemical transformation of yeast cells to increase the resolution of cDNA library screening. MATERIALS AND METHODS: Plasmids pDEST22 and pDEST32 were used for chemical transformation of the yeast strain S. cerevisiae pJ69-4A. For screening, a cDNA library was used, obtained on the basis of mRNA isolated from the roots of pea Pisum sativum cultivar Finale, inoculated with rhizobia. RESULTS: Factors influencing the efficiency of transformation were identified. Among them are the molecular weight of polyethyleneglycol used for transformation, as well as the number of cell division cycles that the culture undergoes. The effect of the number and size of plasmids used in transformation was also shown. Using the optimized protocol, a cDNA library was successfully screened to find coregulators of the NIN transcription factor. CONCLUSIONS: Based on the data obtained, optimal parameters were determined that allow achieving a high level of competence in yeast cells. The use of the described protocol allowed for successful screening of the library to identify coregulators of the NIN transcription factor.
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