Proteolytic fragmentation of polypeptide release factor 1 of Thermus thermophilus and crystallization of the stable fragments

Polypeptide release factor one from Thermus thermophilus, ttRF1, was purifi ed and subjected to crystallization. Thin crystalline needles were obtained but their quality was not satisfactory for X-ray diffraction. Stable fragm ents of ttRF1 suitable for crystallization were screened by limited proteol ysis. Three major fragments were produced by thermolysinolysis and analyzed by N-terminal sequencing and electrospray mass spectrometry. They were N-t erminal fragments generated by proteolysis at amino acid positions 211, 231 and 292. The corresponding recombinant polypeptides, ttRF1(211), ttRF1(231 ) and ttRF1(292), were overproduced and subjected to crystallization Of the se polypeptides, ttRF1292 gave rise to crystals that belong to P3(1) (or P3 (2)) space group with unit cell parameters a = b = 64.5 Angstrom, c = 86.6 Angstrom and diffract up to 7 Angstrom resolution. (C) 2000 Societe francai se de biochimie et biologie moleculaire / Editions scientifiques et medical es Elsevier SAS.