Crystallization of the Fv fragment of mouse myeloma protein M315.

The Fv fragment of mouse myeloma protein M313 was crystallized from poly(ethylene glycol) solution in the form of monoclinic crystals, space group C2 and unit cell dimensions a = 5.96 nm (59.6 A), b = 5.66 nm (56.6 A), c = 13.79 nm (13.9 A) and beta = 99.7 degrees. Some unusual effects of poly(ethylene glycol)on protein crystals were noted and are discussed.     

Preparation of Fv fragment from the mouse myeloma XRPC-25 immunoglobulin possessing anti-dinitrophenyl activity.

The myeloma IgA protein produced by plasmacytoma XRPC-25, was isolated by affinity chromatography on dinitrophenyllysine-Sepharose. The affinity constant of the intact protein or its Fab' toward 2,4-dinitrophenyl-L-lysine (Dnp) was found to be 2.6 X 10(5) M-1. In order to prepare an Fv fragment (Hochman, J., Inbar, D., and Givol, D. (1973), Biochemistry 12, 1130) from this protein, the heavy and light chains were separated and the light chain was digested with trypsin at pH 8.2 to yield half a light chain. This digest was reassociated with the heavy chain and the recombinant was digested with papain at pH 5.7. Fractionation of this digest on a Sephadex G-75 column and Dnp-lysine-Sepharose resulted in the isolation of an Fv fragment which possesses one binding site for Dnplysine (Ka = 2.0 X 10(5) M-1). The active Fv fragment has a molecular weight of 23,400 and is composed of two peptide chains, each having a molecular weight of approximately 12,000. The N-terminal residues of these chains are aspartic and glutamic acids, which are also N-terminal in the heavy and light chains, indicating that the Fv is composed of VL and VH.     

Crystallization and preliminary X-ray diffraction study of a chimaeric Fab' fragment of antibody binding tumour cells

Crystals have been obtained of a chimaeric Fab' fragment that binds to a tumour-associated mucin-like glycoprotein TAG72. The Fab' fragment comprises the variable heavy and light-chain domains of a murine monoclonal antibody, B72.3, coupled to human gamma 4 and kappa constant regions. The crystals are orthorhombic and belong to the space group P2(1)2(1)2(1), with unit cell dimensions a = 67.9 A, b = 94.2 A and c = 208.8 A. Diffraction to 2.6 A resolution was observed using synchrotron radiation. Despite the acute radiation sensitivity of the crystals a full native data set has been collected using the Weissenberg camera at the Photon Factory synchrotron. These data will be used for molecular replacement calculations in an attempt to elucidate the structure of this chimaeric Fab' fragment.     

Symmetry of binding sites of a mouse IgA myeloma protein (MOPC 315)

We have investigated the mechanism of monovalency of the 7S subunit of a mouse IgA myeloma protein (MOPC 315) against a large antigen. This subunit, although it clearly can bind two molecules of a small hapten, fails to precipitate or hemagglutinate the relevant multivalent antigen. In an equilibrium Farr assay, we have shown that the subunit has only one valence for a univalent 40,000 molecular weight antigen (dinitrophenyl-dextran). We have investigated how various levels of affinity labeling quantitatively affect (a) the valence observed in the equilibrium Farr assay against a large antigen, and (b) the binding of the MOPC 315 to an insoluble antigenic matrix. Our results indicate that the Fab regions of the 7S subunit are arranged symmetrically and that the inactivity of one of them toward a large antigen is probably due to steric hindrance caused by the antigen bound to the adjacent site.     

Small-angle neutron scattering studies of the conformation of myeloma protein MOPC315 and its Fab fragment, and the interaction with a monovalent dinitrophenyl hapten

The first small-angle scattering study of an immunoglobulin A is reported. Neutron measurements have been made to determine conformational parameters of the mouse myeloma protein MOPC315 and to relate these to previous immunoglobulin G results. Use of the contrast method shows that the MOPC315 IgA molecule is not simply globular, that it has a dry volume of 220.0 +/- 4.5 nm3 corresponding to a mass density of 1.275 +/- 0.025 g cm-3 and that its full and cross-sectional radii of gyration, corrected for concentration dependence, are 7.97 +/- 0.07 nm, 2.40 +/- 0.08 nm and 1.33 +/- 0.07 nm respectively. Similar study of its Fab fragment gives a dry molecular volume of 69.0 +/- 0.7 nm3, a mass density of 1.285 +/- 0.015 g cm-3 and uncorrected radii of gyration that are consistent with those of the parent and support an overall "T" or "Y" conformation in solution. Addition to saturation of a small monovalent dinitrophenyl hapten leaves the dry volume of the whole molecule unaltered, but may slightly lower one or more of its radii of gyration. The significance of this finding is discussed. Comparative studies with rabbit anti-dinitrophenyl immunoglobulin G antibody suggest a different initial conformation but similar consequences of hapten binding, which, if real, are probably unrelated to classical complement fixation.     

Crystallization and preliminary X-ray analysis of the complex between a mouse Fab fragment and a single IgG-binding domain from streptococcal protein G.

The Fab fragment of a mouse immunoglobulin G1, complexed with a single IgG-binding domain from streptococcal protein G, has been crystallized in a form suitable for analysis by X-ray diffraction. The needle-shaped crystals were grown from polyethylene glycol 4000 using vapour diffusion methods and diffract to 2.3 A resolution. The space group is P2(1)2(1)2(1) (a = 64.5 A, b = 70.5 A and c = 120.1 A), with one Fab-protein G domain complex in the asymmetric unit. Solution of the three-dimensional structure of the complex will permit a detailed analysis of the molecular interactions between protein G and the Fab portion of IgG.     

Crystallization of an anti-2,2,6,6-tetramethyl-1-piperidinyloxy-dinitrophenyl monoclonal antibody Fab fragment with and without bound hapten

The Fab fragment of a monoclonal antibody, AN02, specific for a 2,2,6,6-tetramethyl-1-piperidinyloxy-dinitrophenyl hapten was crystallized both with and without bound hapten. Both crystals formed in phosphate-buffered saline (150 mM-NaCl, 10 mM-Na2PO4, 0.02% (w/v) NaN3 (pH 7.4) at 4 degrees C and diffracted beyond 2.2 A resolution (1A = 0.1 nm). Fab with bound hapten crystallizes in space group P6(1)22 or P6(5)22, with cell dimensions a = b = 73.23 A, c = 373.8 A, alpha = beta = 90 degrees and gamma = 120 degrees. Unoccupied Fab also crystallizes in space group P6(1)22 or P6(5)22 with cell dimensions a = b = 73 A, c = 377 A, alpha = beta = 90 degrees and gamma = 120 degrees.     

Crystallization of a membrane pore-forming protein with mosquitocidal activity from Bacillus thuringiensis subspecies kyushuensis

CytB, a membrane pore-forming toxin from Bacillus thuringiensis subspecies kyushuensis, is specifically toxic to dipteran insect larvae but broadly cytolytic in vitro. It has been purified in the protoxin form from a recombinant Escherichia coli source and crystals have been obtained which diffract X-rays to at least 2.6 Å resolution. The tendency for CytB to aggregate in solution was overcome by including 50 mM of urea or 8 mM of ethanolamine during crystallization. Mutants designed to add or subtract single cysteine residues for the purpose of heavy atom derivative preparation were similarly purified and crystallized. The crystals are hexagonal bipyramids. They belong to space group P6₁22 (or P6₅22) with lattice constants a = b = 67.34 Å, c = 170.96 Å, and contain one molecule of the CytB protoxin (MW 29235) per asymmetric unit and 27% solvent by volume. © 1995 Wiley-Liss, Inc.     

Specificity of interactions of hapten side chains with the combining site of the myeloma protein MOPC 315.

The pKa values of the three histidine residues in the Fv fragment (variable region of the heavy and light chains) of the mouse myeloma protein MOPC 315, measured by high resolution n.m.r. (nuclear magnetic resonance), are 5.9, 6.9 and 8.2. The perturbation of the pKa of one of the histidines (pKa 6.9) on the addition of hapten and the narrow linewidth of its proton resonances suggests that it is at the edge of the combining site. References to the model of the Fv fragment [Padlan, Davies, Pecht, Givol & Wright (1976) Cold Spring Harbor Symp. Quant. Biol. 41, in the press] allows assignment of the three histidine residues, histidine-102H, histidine-97L and histidine-44L. The determination of the pKa of the phosphorus group, by 31P n.m.r., of a homologous series of Dnp- and Tnp- (di- and tri-nitrophenyl) haptens has located a positively charged residue. Molecular-model studies on the conformations of these haptens show that the residue is at the edge of the site. The model suggests that the positively charged residue is either arginine-95L or lysine-52H.     

Crystallization and preliminary x-ray studies of the Fab fragment from a humanized version of the mouse anti-human fas antibody HFE7a

A humanized version of the apoptosis-inducing mouse anti-human Fas monoclonal antibody, HFE7A, is under further development for the treatment of autoimmune diseases such as rheumatoid arthritis. We have crystallized the antigen-binding fragment (Fab) of the humanized HFE7A. The crystals belong to the orthorhombic space group P2(1)2(1)2(1) with cell dimensions a = 54.4 A, b = 82.7 A, c = 104.9 A and contain one Fab molecule in the asymmetric unit. X-ray diffraction data were collected to 2.8 A resolution.