Crystallization and preliminary x-ray diffraction studies of two new antigen-antibody (lysozyme-Fab) complexes

The complexes between the Fab fragments of two monoclonal anti-lysozyme antibodies, Fab10.6.6 (high affinity) and D44.2 (lower affinity), and their specific antigen, hen egg-white lysozyme, have been crystallized. The antibodies recognize an antigenic determinant including Arg68, but differ significantly in their association constants for the antigen. Two crystalline forms were obtained for the complex with FabF10.6.6, the higher affinity antibody. One of them is monoclinic, space group P21, with unit cell dimensions a = 145.6 A, b = 78.1 A, c = 63.1 A, beta = 89.05 degrees, consistent with the presence of two molecules of the complex in the asymmetric unit. These crystals diffract X-rays beyond 3 A making this form suitable for high-resolution X-ray diffraction studies. The second form crystallizes in the triclinic space group P1, with unit cell dimensions a = 134.0 A, b = 144.7 A, c = 98.6 A, alpha = 90.30 degrees, beta = 97.1 degrees, gamma = 90.20 degrees, consistent with the presence of 10 to 12 molecules of the complex in the unit cell. These crystals do not diffract X-rays beyond 5 A resolution. The antigen-antibody complex between FabD44.2, the lower affinity antibody, and hen egg-white lysozyme crystallizes in space group P2(1)2(1)2(1), with unit cell dimensions a = 99.7 A, b = 167.3 A, c = 84.7 A, consistent with the presence of two molecules of the complex in the asymmetric unit. These crystals diffract X-rays beyond 2.5 A resolution.     

Structure determination of a monoclonal Fab fragment specific for histidine-containing protein of the phosphoenolpyruvate: sugar phosphotransferase system of Escherichia coli

Jel 42 is a monoclonal antibody specific for histidine-containing protein, a small phosphocarrier protein required for sugar transport in Escherichia coli. Fab fragments prepared from this antibody by papain digestion consisted of three major isoelectric forms which were separated on a chromatofocusing column. Two of these forms produced large crystals in space group P21 and unit cell dimensions a = 117.48 A, b = 66.56 A, c = 67.31 A, and beta = 118.7 degrees, with two Fab fragments per asymmetric unit. Data were collected to 3.5-A resolution. The structure of Fab Jel 42 was solved by the Molecular Replacement method (least-squares refined to R = 0.282) using the known structure of Fab HED 10 (12) as the search model; the amino acid residues of the hypervariable and elbow regions of Fab HED 10 were omitted from the starting model. A Fourier map calculated at this stage revealed electron density which corresponded to the hypervariable loops forming the antigen-binding crevice and the elbow region of Fab Jel 42. The elbow angles for the two independent Fab molecules are 159 and 167 degrees, similar to that of the Fab HED 10 search model which has an elbow angle of 162 degrees. There is no local noncrystallographic axis of symmetry relating the two molecules in the asymmetric unit.     

Crystallization and preliminary x-ray diffraction studies of the Fab fragment of a neutralizing monoclonal antibody directed against human rhinovirus serotype 2

We report on the preparation, crystallization, and preliminary x-ray diffraction analysis of the Fab fragment of the monoclonal antibody 8F5 that neutralizes infectivity of human rhinovirus serotype 2 (HRV2). Fab fragments prepared from this antibody by papain digestion were purified to isoelectric homogeneity by ion exchange chromatography and chromatofocusing. Crystals were obtained by the hanging drop vapor diffusion method using ammonium sulfate as precipitant. The crystals belong to the orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions a = 59.9 A, b = 86.3 A, c = 128.2 A and diffract to at least 2.8-A resolution. The cell volume suggests the presence of one molecule per asymmetric unit, and the solvent content is estimated to be 61%.     

Preliminary crystallographic study of the Fab fragments of two monoclonal anti-2-phenyloxazolone antibodies

We report on the preparation, crystallization, and preliminary x-ray crystallographic study of the Fab fragments of two monoclonal anti-2-phenyloxazolone antibodies obtained from the secondary response to this hapten. The Fab fragment from one of these (NQ10/12.5) has been crystallized from polyethylene glycol 8000 solutions in a form suitable for high-resolution x-ray crystallographic studies. These crystals are monoclinic, space group C2, with a = 129.2 A, b = 79.4 A, c = 57.7 A, beta = 96.2 degrees, and one Fab/asymmetric unit. Determination of the three-dimensional structure of Fab NQ10/12.5 should help clarify the role of somatic mutation in the maturation of an immune response. This antibody and an anti-lysozyme antibody also under study apparently use the same germ-line encoded VK and a similar VH gene, respectively, as the idiotypic anti-oxazolone antibodies characteristic of the primary response. A comparative study of the two structures should shed light on the role of the pairing of heavy and light chains in the antigen-binding function of antibodies.     

Preliminary crystallographic study of a complex between the Fab fragment of a monoclonal anti-lysozyme antibody (D1.3) and the Fab fragment from an anti-idiotopic antibody against D1.3

An anti-lysozyme antibody, D1.3, was used as immunogen to obtain syngeneic (Balb/c) monoclonal anti-idiotopic antibodies. The complex between Fab D1.3 and the Fab fragment from the anti-idiotopic antibody E225 has been crystallized. The crystals are monoclinic, space group P2(1), with a = 75.7 A, b = 77.4 A, c = 97.2 A, beta = 111.90 degrees and one molecule of the complex in the asymmetric unit. X-ray photographs show reflections extending to a resolution of about 3 A. Although twinning occurs frequently in the large crystals obtained, this material is suitable for high-resolution X-ray analysis.     

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.     

Crystal parameters and molecular replacement of an anticholera toxin peptide complex.

TE33 is an Fab fragment of a monoclonal antibody raised against a 15-residue long peptide (CTP3), corresponding in sequence to residues 50-64 of the cholera toxin B subunit. Crystals of the complex between TE33 and CTP3 have been grown from 20% (w/v) polyethylene glycol-8000 at pH 4.0. The crystals are orthorhombic, space group P2(1)2(1)2, with unit cell dimensions a = 104.15, b = 110.61, and c = 40.68 A. X-Ray data have been collected to a resolution of 2.3 A. The asymmetric unit contains one molecule of Fab and one molecule of CTP3. The presence of CTP3 has been demonstrated by fluorescence quenching of the dissolved crystal after X-ray data collection. A molecular replacement solution was found based on the coordinates of DB3, an antiprogesterone Fab fragment.     

Crystallization of the Fab from a human monoclonal antibody against gp 41 of human immunodeficiency virus type I

A monoclonal IgG antibody directed against gp 41 from the human immunodeficiency virus (HIV-1) has been crystallized in both intact and Fab forms. Crystals of the intact antibody grow as tetragonal-like prisms too small for conventional X-ray analysis. However, the Fab portion of the antibody produces suitable plate-like crystals which belong to the space group P2(1)2(1)2(1) with unit cell constants of a = 66.5 A, b = 74.3 A and c = 105.3 A. There is one molecule of Fab in the asymmetric unit. The Fab crystals show diffraction to d-spacings less than 3.0 A.     

Two routes for production and purification of Fab fragments in biopharmaceutical discovery research: Papain digestion of mAb and transient expression in mammalian cells

Fab (fragment that having the antigen binding site) of a monoclonal antibody (mAb) is widely required in biopharmaceutical research and development. At Centocor, two routes of Fab production and purification were used to enable a variety of research and development efforts, particularly, crystallographic studies of antibody–antigen interactions. One route utilizes papain digestion of an intact monoclonal antibody for Fab fragment production. After digestion, separation of the Fab fragment from the Fc (fragment that crystallizes) and residual intact antibody was achieved using protein A affinity chromatography. In another route, His-tagged Fab fragments were obtained by transient expression of an appropriate construct in mammalian cells, and typical yields are 1–20 mg of Fab fragment per liter of cell culture. The His-tagged Fab fragments were first captured using immobilized metal affinity chromatography (IMAC). To provide high quality protein sample for crystallization, Fabs from either proteolytic digestion or from direct expression were further purified using size-exclusion chromatography (SEC) and/or ion-exchange chromatography (IEC). The purified Fab fragments were characterized by mass spectrometry, SDS–PAGE, dynamic light scattering, and circular dichroism. Crystallization experiments demonstrated that the Fab fragments are of high quality to produce diffraction quality crystals suitable for X-ray crystallographic analysis.     

Preliminary crystallographic study of the Fab fragment of a monoclonal antibody directed against a cobra cardiotoxin

We report on the preparation, crystallization and preliminary X-ray crystallographic study of the Fab fragments from a murine monoclonal anti-cardiotoxin antibody M gamma 2-3 directed against a cobra cardiotoxin. The Fab fragment has been crystallized from polyethylene glycol 8000 solutions in a form suitable for high-resolution, X-ray crystallographic studies. The crystals are monoclinic, space group C2, with a = 161.2 A, b = 40.4 A, c = 96.5 A, beta = 118.3 degrees.     

Conformational flexibility and crystallization of tandemly linked type III modules of human fibronectin.

Fibronectin is a large cell adhesion molecule that is composed of several functional domains. The cell-binding domain that binds to cell surface integrins consists of repeated homologous type III modules. In this study, recombinant fragments from the cell-binding domain of human fibronectin that participate in a newly characterized fibronectin-fibronectin interaction with FNIII1 were crystallized. In each case, the crystals had more than one fibronectin fragment in the asymmetric unit. Crystals of FNIII10-11 grew in the space group C2 with a = 117.1 A, b = 38.6 A, c = 80.6 A, beta = 97.2 degrees, and two molecules in the asymmetric unit. These crystals diffracted to 2.5 A resolution. Fragment FNIII8-11 and a shorter fragment, FNIII8-10, crystallized in hexagonal space groups with large unit cells and two to four molecules per asymmetric unit. Even very large crystals of these fragments did not diffract beyond 4 A. The crystal packing for this collection of fibronectin fragments suggests conformational flexibility between linked type III modules. The functional relevance of this flexibility for elongated versus compact models of the cell-binding domain of fibronectin is discussed.     

Crystallization and preliminary X-ray studies of the VL domain of the antibody McPC603 produced in Escherichia coli

The VL domain, obtained from a recombinant Fv fragment of the antibody McPC603 expressed in Escherichia coli, has been crystallized as a dimer from 2 M-(NH4)2SO4 (pH 4.0). The crystals are hexagonal, space group P6(1)22. The cell dimensions are a = b = 86.48 A, c = 76.64 A, with a VL monomer as the asymmetric unit. The crystals diffract to 2.0 A. The structure was solved by Patterson search using the VL domain of the Fab fragment of McPC603 and the VL dimer REI.     

Characterization of crystals of an intact monoclonal antibody for canine lymphoma

A monoclonal antibody of the subclass IgG2a specific for canine lymphoma cells has been crystallized by vapor diffusion from polyethylene glycol 8000. the crystals, which occasionally measure nearly a millimeter on edge, have been examined by X-ray diffraction. The crystals are of triclinic space group P1 with unit cell parameters of a = 66.39 A, b = 77.34 A, c = 101.42 A, alpha = 87.60 degrees, beta = 92.55 degrees, gamma = 97.54 degrees and cell volume of V = 4.84 x 10(5) A3. There is one entire antibody molecule as the asymmetric unit of the crystals. Three-dimensional X-ray diffraction data have been collected to 2.8 A resolution and a self rotation function calculation shows a pronounced peak indicating at least an approximate non-crystallographic dyad axis.     

Preparation, crystallization and preliminary X-ray analysis of the Fab fragment of monoclonal antibody MN423, revealing the structural aspects of Alzheimer's paired helical filaments

Monoclonal antibody (mAb) MN423 recognizes Alzheimer's disease specific conformation of tau protein assembled into paired helical filaments (PHF). Since the three-dimensional structure of PHF is currently unavailable, the structure of MN423 binding site could provide important information about PHF conformation with the consequences for the Alzheimer's disease prevention and cure. Fab fragment of MN423 was prepared and purified. We have identified two different conditions for crystallization of the Fab fragment that yielded two crystal forms. They diffracted to 3.0 and 1.6 A resolution with four and one molecule in the asymmetric unit, respectively. Both crystal forms belonged to the space group P2(1) with unit cell parameters a = 76.4 A, b = 138.4 A, c = 92.4 A, beta = 101.9 degrees , and a = 71.5 A, b = 36.8 A, c = 85.5 A, beta = 113.9 degrees .     

Crystallization and preliminary x-ray diffraction studies of a monoclonal antibody fab fragment against foot-and-mouth disease virus and of its complex with the main antigenic site peptide

The Fab fragment of the neutralizing monoclonal antibody SD6 elicited against foot-and-mouth disease virus (FMDV) C-SBcl and its complex with a peptide, corresponding to the major antigenic site of FMDV (VPl residues 136–150, YTASARGDLAHLTTT), have been crystallized using the hanging drop vapor diffusion techniques. For the isolated Fab, crystals diffracting to 2.5 Å resolution were obtained at room temperature using ammonium sulfate as precipitant. These crystals are monoclinic, space group C2, and unit cell parameters a = 109.53 Å, b = 89.12 Å, c = 64.04 Å, and β = 112.9° and contain one Fab molecule per asymmetric unit. Crystals from the complex diffract, at least, to 2.8 Å resolution and were obtained, at room temperature, using PEG as precipitant. These crystals are monoclinic, space group P2, and unit cell parameters a = 56.11 Å, b = 60.67 Å, c = 143.45 Å, and β = 95.4°, Density packing considerations indicate that there are two Fab molecules in the asymmetric unit. © 1994 John Wiley & Sons, Inc.     

Crystallization of a soluble form of the rat T-cell surface glycoprotein CD4 complexed with Fab from the W3/25 monoclonal antibody

The structure of the T-lymphocyte cell surface glycoprotein CD4 is of considerable biological and medical interest. Recombinant rat CD4 expressed in soluble form in mammalian cells and complexed with W3/25 monoclonal Fab fragments formed crystals that diffract to 3.5 A and have the orthorhombic space group P2(1)2(1)2 or P2(1)2(1)2(1). The unit cell has dimensions a = 317 A, b = 161 A and c = 41.8 A and the asymmetric unit consists of two CD4:Fab complexes. These crystals are of suitable quality for X-ray diffraction analysis.     

Optimization of crystals of an inhibitory antibody of urokinase plasminogen activator receptor (uPAR) with hydrogen peroxide and low protein concentration

Optimization of protein crystal formation is often a necessary step leading to diffraction-quality crystals to enable collection of a full X-ray data set. Typical protein crystal optimization involves screening different components, e.g., pH, precipitants, and additives of the precipitant solution. Here we present an example using an inhibitory antibody of urokinase plasminogen activator receptor (uPAR) where such procedures did not yield diffracting crystals. In contrast, it was the treatment of the protein with hydrogen peroxide incubation and the protein concentration reduction that were found to be key factors in obtaining diffracting crystals. Final crystals diffracted to 1.75 A, and belong to orthorhombic P2(1)2(1)2(1) space group with unit cell parameters a = 37.162 A, b = 84.474 A, c = 134.030 A, and contain one molecule of Fab fragment of anti-uro kinase receptor antibody in the asymmetric unit.     

Crystallization of the complex of a monoclonal Fab fragment with the histidine-containing protein of the phosphoenolpyruvate: sugar phosphotransferase system of Escherichia coli

Single crystals of the complex of a monoclonal Fab fragment with the histidine-containing protein of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli have been grown. This represents one of the first Fab-protein antigen complexes in which the same Fab fragment has previously been crystallized in the uncomplexed state and the structure solved (Prasad, L., Vandonselaar, M., Lee, J. S., and Delbaere, L. T. J. (1988) J. Biol. Chem. 263, 2571-2574). Single crystals up to 0.25 x 0.50 x 0.05 mm in size were grown by the technique of washing and reseeding. The space group is C2, with unit cell dimensions a = 130.0, b = 68.1, and c = 77.6 A; beta = 97.3 degrees; and Z = 4. There is one Fab-histidine-containing protein complex/asymmetric unit, and the solvent content is estimated to be 57%.     

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.     

Biochemical implications from the variable gene sequences of an anti-cytochrome c antibody and crystallographic characterization of its antigen-binding fragment in free and antigen-complexed forms.

To study the nature of antibody-antigen interactions, we have determined the variable gene sequences of the anti-cytochrome c immunoglobulin G1 (IgG1) monoclonal antibody E8, and obtained diffraction-quality crystals of the E8 antigen-binding fragment (Fab), both free and bound to its antigen, horse cytochrome c. The FabE8 crystals belong to space group P21 with unit cell dimensions of a = 45.0 A, b = 85.1 A, c = 63.3 A and beta = 105.5 degrees, have one FabE8 molecule per asymmetric unit and diffract to at least 2.1 A resolution. Crystals of the FabE8-cytochrome c complex belong to space group P212121 with unit cell dimensions of a = 84.3 A, b = 73.3 A and c = 94.9 A, accommodate one complex per asymmetric unit and diffract to 2.4 A resolution. In the nucleotide-derived amino acid sequences, the light-chain variable domain (VL) but not the heavy-chain variable domain (VH) of E8 is nearly identical to that of the anti-lysozyme antibody D1.3, differing by only five amino acid residues. Only one of these interacts with lysozyme in the D1.3-lysozyme crystal structure. Six negative and four positive charges in the VH complementarity determining regions of E8 complement four positive and three negative charges in the E8 epitope on cytochrome c. These data suggest that only a subset of the residues in an antibody-protein interface may be critical for binding and that the VH may play a dominant role in antigenic recognition.