Transcription activator-like effectors (TALEs) are proteins widely used in synthetic biology because they can bind to defined binding sites in the polynucleotide chain. They consist of many short amino acid sequence repeats (the so-called modules or canonic repeats of 33 to 35 residues). TALE specificity can be regulated by changing two amino acid residues in the modules that recognize and bind to consecutive nucleotides on one of the DNA strands. In addition to splicing with effector domains and DNA editing, they can influence gene transcription triggered by another transcription factor. To evaluate the binding affinity of the transcription factor TetR with simultaneous binding of the TALE [A] protein to DNA, we expressed both proteins in Escherichia coli. They were isolated by immobilized metal affinity chromatography and further purified by gel filtration. To optimize the purification, we tested several different chromatographic buffers. Using analytical gel filtration coupled to multi-angle laser light scattering (SEC-MALS), dynamic light scattering techniques, and polyacrylamide gel electrophoresis in the presence of sodium lauryl sulfate, we demonstrated that the experimentally determined sizes and molecular masses were comparable to the theoretical values and that the proteins did not interact with each other. Using the electrophoretic mobility shift assay (EMSA), we determined the dissociation constant (Kd) of the TetR:DNA complex. However, we could not determine the binding affinities of TALE [A] to DNA and that of TetR to DNA in the presence of TALE [A] because the binding of TALE [A] to DNA was inconsistent. The inconsistency could be due to the different conformational states of the recombinant TALE [A].
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