Figure 2.

Insertion of ncaa and bio-orthogonal labeling of RPA. (A) Plasmids used for the overexpression of RPA and ncaa components. The three subunits of RPA are cloned into a pET vector and the RPA32 subunit is engineered to carry a C-terminal polyhistidine tag and a TAG inserted for the incorporation of 4AZP. The genes for the tRNA that recognizes the amber suppressor codon and inserts 4AZP (tRNA_CUA) and its corresponding tRNA synthetase are engineered into the pDULE2-pCNF plasmid. (B) SDS-PAGE analysis of RPA^WT, RPA^4ZAP and the MB543-labeled RPA (RPA^f)proteins are shown after coommassie staining (left) or fluorescence imaging (right). Site-specific fluorescence labeling of the RPA32 subunit is observed.