Purification and cloning of DNA binding transcription factors

Abstract

Abstract If you have just identified a protein binding to a novel regulatory sequence, the next step is to clone the cDNA encoding the protein. Most practitioners in this field would first attempt direct screening of expression libraries with the DNA binding site (see Chapter 5) before embarking on the protein purification/peptide sequence/oligonucleotide screening approach. However, in many cases, direct screening of libraries has not proved successful or has not been possible (for example when the protein is part of a multiprotein complex and more than one protein is required for DNA binding) and protein purification may then have been the only method available. The purification of DNA binding proteins has been remarkably successful with the development of sequence-specific DNA affinity chromatography and the band-shift technique for rapidly analysing chromatography fractions. With the recent advances in protein-sequencing techniques (e.g. sequencing peptides immobilized on blots following electrophoretic separation), the protein purification approach has become less arduous and one can expect to get good sequence results from 50 pmol of protein. Also, mass spectroscopy techniques are being used to mass fingerprint proteins for the identification of protein sequences in databases, and tandem mass spectroscopy is now able to obtain peptide sequences from femtomole amounts of protein.