Expression, purification, crystallization and preliminary crystallographic analysis of a stand-alone RAM domain with hydrolytic activity from the hyperthermophile Pyrococcus furiosus.

ABSTRACT: The RAM domain is one of several ligand-binding modules present in prokaryotes that are presumed to regulate the transcription of specific genes. To date, no hydrolytic activity has been reported for such modules. Curiously, a stand-alone RAM domain in Pyrococcus furiosus was isolated during a screen for hydrolytic activity against chromogenic esters. The gene encoding this protein was cloned and expressed in Escherichia coli and crystallized after a single purification step. X-ray diffraction data from the crystals were obtained to a resolution of 2.8 A using a conventional X-ray source. The cocrystallization of the recombinant protein with 1,2-epoxy-3-(4-nitrophenoxy)propane (EPNP) and phenylmethylsulfonyl fluoride (PMSF) produced crystals that yielded data to 2.2 and 2.8 A, respectively

SUBMITTER: Agapay RC 

PROVIDER: S-EPMC1991312 | biostudies-literature | 2005 Oct

REPOSITORIES: biostudies-literature