| Abstract [eng] |
Protein in vitro evolution is one of the most powerful methods for modifying protein properties. In this dissertation, a newly developed compartmentalized ribosome display (CRD) method was employed. CRD combines ribosome display with the compartmentalization technique, enabling free interaction between the protein and its substrate while maintaining the essential genotype–phenotype linkage through a physical barrier. The CRD method was applied to the directed evolution of the M-MuLV reverse transcriptase. After selection for thermostable M-MuLV RT mutants, several previously unknown mutations enhancing the protein resistance to increased temperatures were identified. Rationally designed multiple mutants, generated by combining the most beneficial point mutations, exhibited significantly increased substrate-binding affinity and processivity and were able to synthesize cDNA at elevated temperatures. A subsequent experiment focused on selecting mutants capable of efficiently synthesizing cDNA at low dNTP concentrations. This selection yielded several active-site mutants with higher affinity to the dNTP substrate. Taken together, the results of these selection experiments demonstrate the potential of the CRD method for evolving enzymes with desired properties. Notably, the outcomes of the in vitro selection experiments are consistent with principles observed in natural evolution. |
