| Abstract [eng] |
The goal of this work was to create yeast protein Sup35 mutant variant K102R and to estimate its synthesis in cells of S. cerevisiae 74D-694 [psi-][PIN+]. In order to achieve this, primers were created and site-directed mutagenesis was used to change one nucleotide in the sequence through three independent PCR reactions. Ist and IInd reaction products were AB (332 bp) and CD (2521 bp), IIIrd reaction product – those two fragments fused into one AD fragment (SUP35K102RGFP) (2827 bp). Later this fragment was inserted into pJET1.2/blunt cloning vector and successfully multiplied in Escherichia coli DH5α cells. After that mutated gene was inserted into yeast shuttle vector pRSCup and multiplied in E. coli DH5α cells and in Saccharomyces cerevisiae cells. SUP35K102RGFP was sequenced after every step of the research and analyzed using Benchling online application which showed that creation of yeast gene SUP35 mutant variant K102R was achieved. Then, Sup35K102RGFP and Sup35GFP protein synthesis was induced by copper ions in the S. cerevisiae cells, all yeast cell proteins were purified by alkaline lysis and their analysis was performed in polyacrylamide gel with sodium dodecyl sulfate (SDS-PAGE). Synthesis of Sup35K102RGFP and Sup35GFP in the S. cerevisiae 74D-694 [psi-][PIN+] strain was not evaluated due to inefficient induction of the CUP1 promoter and/or incomplete purification of all the proteins of the cell or alkaline lysis was not enough sensitive method in this research. |
