[29] Wild-type and hexahistidine-tagged derivatives of leucine-responsive regulatory protein from escherichia coli

Leucine-responsive regulatory protein (Lrp) is a moderately abundant regulatory protein in Escherichia coli that stimulates expression of more than a dozen operons and represses expression of another dozen. Proteins having nearly identical amino acid sequences exist in other enteric bacteria and regulatory proteins that are clearly evolutionarily related to Lrp are found in a large number of microorganisms. The acronym Lrp derives from the fact that leucine, when present in high concentrations in media, can modulate the effect of Lrp. In many cases, leucine has little or no effect on Lrp-mediated expression in vivo, but may show small effects in vitro. In some well-documented instances, exogenous leucine reduces the effect of Lrp, whereas in other instances it potentiates the effect of Lrp. Alanine also modulates the effect of Lrp on some promoters. This chapter describes the purification of wild-type Lrp and of a hexahistidine (His6)-tagged Lrp that is easy to purify and that is useful for a variety of in vitro experiments. The His6 derivative behaves like the wild-type protein in a number of ways, both in vitro and in vivo.