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 purified and subjected to crystallization. Thin crystalline needles were obtained but their quality was not satisfactory for X-ray diffraction. Stable fragments of ttRF1 suitable for crystallization were screened by limited proteolysis. Three major fragments were produced by thermolysinolysis and analyzed by N-terminal sequencing and electrospray mass spectrometry. They were N-terminal 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 these polypeptides, ttRF1(292) gave rise to crystals that belong to P3(1) (or P3(2)) space group with unit cell parameters a = b = 64. 5 A, c = 86.6 A and diffract up to 7 A resolution.
Tin
O F
OF
Institute of Protein Research, Russian Academy of Sciences, Moscow Region, Pushchino, Russia.
Rykunova
A I
AI
Muranova
T A
TA
Toyoda
T
T
Itoa
K
K
Suzuki
T
T
Watanabe
K
K
Garber
M B
MB
Nakamura
Y
Y
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry*; D003001:Cloning, Molecular; D003460:Crystallization; D004926:Escherichia coli; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000302:isolation & purification; D010454:Peptide Termination Factors / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013820:Thermolysin; D016963:Thermus thermophilus / Q000235:genetics / Q000378:metabolism*; D015534:Trans-Activators / Q000737:chemistry*
D001426:Bacterial Proteins; D010446:Peptide Fragments; D010454:Peptide Termination Factors; D011994:Recombinant Proteins; D015534:Trans-Activators; C041460:peptide chain termination release factor 2; D013820:Thermolysin