Expression, purification, crystallization and preliminary X-ray diffraction studies of bacterial and archaeal L4 ribosomal proteins.
Ribosomal protein L4 is implicated in the peptidyltransferase activity of the ribosome and in certain bacteria it regulates the transcription and translation of the 11-gene S10 operon. The genes for the L4 ribosomal proteins from the hyperthermophilic bacterium Thermotoga maritima and the halophilic archaeon Haloarcula marismortui have been PCR amplified from genomic DNA and cloned under the control of a T7 promoter to generate overexpressing Escherichia coli strains. For both proteins, efficient purification procedures were developed to yield material suitable for crystallization trials. Crystals of T. maritima L4 were obtained in the orthorhombic space group P2(1)2(1)2(1), with one molecule per asymmetric unit, diffracting to 1.7 A resolution with synchrotron radiation. Crystals of H. marismortui L4 belonged to the trigonal space group P3(1)21 or P3(2)21 and diffracted to 3.2 A resolution with a rotating-anode source, presumably containing three molecules per asymmetric unit. The results demonstrate that for certain halophilic proteins the same purification and crystallization procedures can be employed as for conventional proteins.
Worbs
M
M
Max-Planck-Institut für Biochemie, Abteilung Strukturforschung, Am Klopferspitz 18a, D-82152 Martinsried, Germany.
Wahl
M C
MC
D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray / Q000379:methods; D019614:Haloarcula marismortui / Q000235:genetics; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D012269:Ribosomal Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D020124:Thermotoga maritima / Q000235:genetics
D019843:Archaeal Proteins; D001426:Bacterial Proteins; D011994:Recombinant Proteins; D012269:Ribosomal Proteins; C041174:ribosomal protein L14