TY - JOUR
T1 - Crystallization of immunoglobulin Fab fragments specific for DNA
AU - Boodhoo, A.
AU - Mol, C. D.
AU - Lee, J. S.
AU - Anderson, W. F.
N1 - Copyright:
Copyright 2004 Elsevier B.V., All rights reserved.
PY - 1988
Y1 - 1988
N2 - The purification and crystallization of Fab fragments of two mouse monoclonal immunoglobulins specific for different DNA structures are described. In each case, papain digestion of the immunoglobulins produced a mixture of Fab species differing in their isoelectric points. Purification of one of these species was required to obtain suitable crystals. One of these antibodies, Jel 72, is specific for right-handed duplex poly(dG)·poly(dC). An Fab fragment of Jel 72 with a pI of 8.8 was purified by anion-exchange chromatography and used to obtain crystals from 56% saturated ammonium sulfate and 50 mM sodium acetate, pH 4.2, that diffract to 2.6-Å resolution. They belong to the orthorhombic space group P212121, with cell dimensions of a = 94.6, b = 102.6, c = 92.4 Å. The other antibody, Jel 318, binds triple-stranded DNA poly[d(Tm5C)]·poly[d(GA)]·poly[d(m5C+T)]. Jel 318 Fab fragments with isoelectric points of 7.6 and 7.8 were also purified by anion-exchange chromatography, and crystals were obtained from 12% polyethylene glycol 8000, 50 mM NaCl, and 10 mM Tris·HCl, pH 7.8. These crystals diffract to about 2.4-Å resolution and also belong to the orthorhombic space group P212121, with cell dimensions of a = 82.4, b = 139.5, and c = 42.0 Å. For both Fab fragments, crystal size and quality improved dramatically upon purification of an individual isoelectric species.
AB - The purification and crystallization of Fab fragments of two mouse monoclonal immunoglobulins specific for different DNA structures are described. In each case, papain digestion of the immunoglobulins produced a mixture of Fab species differing in their isoelectric points. Purification of one of these species was required to obtain suitable crystals. One of these antibodies, Jel 72, is specific for right-handed duplex poly(dG)·poly(dC). An Fab fragment of Jel 72 with a pI of 8.8 was purified by anion-exchange chromatography and used to obtain crystals from 56% saturated ammonium sulfate and 50 mM sodium acetate, pH 4.2, that diffract to 2.6-Å resolution. They belong to the orthorhombic space group P212121, with cell dimensions of a = 94.6, b = 102.6, c = 92.4 Å. The other antibody, Jel 318, binds triple-stranded DNA poly[d(Tm5C)]·poly[d(GA)]·poly[d(m5C+T)]. Jel 318 Fab fragments with isoelectric points of 7.6 and 7.8 were also purified by anion-exchange chromatography, and crystals were obtained from 12% polyethylene glycol 8000, 50 mM NaCl, and 10 mM Tris·HCl, pH 7.8. These crystals diffract to about 2.4-Å resolution and also belong to the orthorhombic space group P212121, with cell dimensions of a = 82.4, b = 139.5, and c = 42.0 Å. For both Fab fragments, crystal size and quality improved dramatically upon purification of an individual isoelectric species.
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M3 - Article
C2 - 3192550
AN - SCOPUS:0024224568
SN - 0021-9258
VL - 263
SP - 18578
EP - 18581
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 34
ER -