Purification and Labeling of Recombinant RecA Proteins

His-tagged pol V was purified from E. coli stain RW644 as described by Karata et al. (2012) (link) and RecA WT was purified by a standard protocol (Cox et al., 1981 (link)). RecA E38K/K72R and RecA E38K/ΔC17 were provided by Michael Cox at the University of Wisconsin, Madison. We incorporated p-azido-L-phenylalanine (pAzF) (Chin et al., 2002 (link)) into RecA WT to site specifically label the protein with Alexa Fluor 488 DIBO alkyne. For the cloning of RecA, we used the pAIR79 plasmid, which was a gift from Michael Cox at the University of Wisconsin, Madison. The Phe21 sequence in RecA WT was replaced with the amber codon via site-directed mutagenesis using Pfu Ultra polymerase (Agilent Technologies). Once the sequence was confirmed, pAIR79 was cotransformed with the vector pEVOL-pAZF (a gift from the Peter Schultz lab at The Scripps Research Institute, San Diego, CA) into the BLR expression strains (Young et al., 2010 (link)). The RecA_F21AzF protein was purified using the same standard protocol for RecA WT (Cox et al., 1981 (link)). RecA_F21AzF was labeled with Alexa Flour 488 (RecA_F21AzF-Alexa Fluor 488) according to manufacturer's instructions (Life Technologies).