EXPRESSION, PURIFICATION, AND FUNCTIONAL-ANALYSIS OF THE TYRR PROTEINOF HAEMOPHILUS-INFLUENZAE

The gene that was inferred to encode the TyrR protein of Haemophilus i nfluenzae Rd was synthesized by polymerase chain reaction and inserted into a T7-based expression vector. Methods were developed to overexpr ess the TyrR protein of H. influenzae in Escherichia coil and to purif y the protein on a large scale. Both in vitro and in vivo functional c omparisons of the H. influenzae and E. coil TyrR proteins were carried out. The TyrR protein of H. influenzae was able to bind in vitro to a n operator target upstream of the aroF-tyrA gene of E. coil. In the pr esence of [gamma-S]ATP, the DNA binding ability of the H. influenzae T yrR protein was drastically reduced. Despite the much shorter peptide chain length (318 amino acid residues vs 513), the TyrR protein of H. influenzae was as active in repressing the aroF promoter as the TyrR p rotein off. coil. Repression by both proteins was enhanced in the pres ence of tyrosine; however, the transcriptional activation function ass ociated with the TyrR protein of E. coil could not be detected when th e H. influenzae TyrR protein was expressed in E. coil. By computer ana lysis, at least five operator targets for TyrR were identified within the genomic DNA of H. influenzae. These observations show that the ass ignment of function to the tyrR gene of H. influenzae was correctly ma de. Further studies of the H. influenzae TyrR protein in comparison to its E. coil counterpart should provide valuable mechanistic informati on on transcriptional regulation in this system. (C) 1997 Academic Pre ss.