Abstract
Crystals of tumor necrosis factor (TNF) have been obtained in two forms. Rhombohedral crystals grow in 1.8 to 2.0 M ammonium sulfite, pH 7.8 at 21 degrees C, and tetragonal crystals grow in 2.6 M magnesium sulfate, pH 5.5 at 25 degrees C. Analysis of TNF by isoelectric focusing under native and denaturing conditions indicates that TNF molecules exist as trimers in solution. The rhombohedral cachectin crystals belong to space group R3 and have unit cell constants a = b = c = 47.65 A and alpha = beta = gamma = 88.1 degrees. Density determinations and the space group indicate that the unit cell contains one 51,000-dalton trimer. These crystals are stable in the x-ray beam and diffract to at least 1.85 A but are apparently twinned by merohedry. The tetragonal crystals are space group P4(3)2(1)2 or its enantiomorph P4(1)2(1)2 and have unit cell constants a = b = 95.08, c = 117.49. The asymmetric unit contains one trimer; the crystals are stable in the x-ray beam and diffract to beyond 3 A.
Highlights
Matureprotein (157 residues, M, = 17,000) is derived by cleavage of the 76 amino-terminal residues from the prohormone, contains two cysteines believed to form an intrachain disulfide bond, and is not glycosylated
Under native conditions tumor necrosis factor (TNF) has been reported to exist in oligomeric states with
Rhombohedralcrystals grow in quaternary structure of TNF in solution
Summary
The sequence of mature TNF is 28% identical to that of Recombinant Human Tumor Necrosis Factor (Cachectin)". Crystals of tumor necrosis factor (TNF) have been nation and describe experiments that confirm the trimeric obtained intwo forms. 1.8 to 2.0 M ammonium sulfite, pH 7.8 at 21 "C, and tetragonal crystals grow in 2.6 M magnesium sulfate, MATERIALS ANDMETHODS pH 5.5 at 25 "C.Analysis of TNFby isoelectric focus- TNF was produced from a synthetic construcwt, hich was expressed ing under native and denaturing conditions indicates asan intracellular protein in yeast. Tumor necrosis factor (cachectin) is a metabolically active protein secreted by macrophages in response to certain invasive stimuli, in response to challenge with endorate of 0.5 ml/min with a solution containing 10 ml of Polybuffer 74TM/100ml of H20, pH5.0. Isoelectric focusing; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diolS;DS, sodium dodecyl sulfate, PAGE, RESULTSANDDISCUSSION polyacrylamide gel electrophoresis; ODD-IEF, one dimension denatured isoelectric focusing; BES, N,N-bis(2-hydroxyethyl)-2-amino- Characterization-Purified TNF migrates as asingle sharp ethanesulfonic acid; MES, 2-(N-morpholino)ethanesulfonicacid.
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