| Abstract [eng] |
Bacteriophage T7 RNA polymerase (T7 RNAP) is an enzyme that catalyses transcription reaction and is widely used tool in molecular biology for RNA synthesis in vivo and in vitro. During in vitro transcription in addition to the main RNA transcript, there are also by-products such as unincorporated nucleotides, abortive or partially synthesised transcripts that are formed and can be removed by optimising DNA template or during purification step. Other major by-product are double-stranded RNA (dsRNA) molecules that are more difficult to remove during purification. It was previously demonstrated that the level of such dsRNA by-products can be lowered by performing in vitro transcription at elevated temperatures which requires usage of thermostable T7 RNAP. During this research properties of potentially thermostable point mutants of T7 RNAP were investigated such as their ability to perform in vitro transcription at elevated temperatures. Variants demonstrating improved properties were selected for protein purification and further experiments for determining their thermostability and the level of dsRNA that is formed during in vitro transcription in different temperatures. Mutations showing the largest improvement in increasing polymerase thermostability were combined into twofold mutants for investigation of mutation additivity and their ability to carry out in vitro transcription at elevated temperature. |
