J. Mol. Biol. (1990) 216, 511-512
Crystallization of the Fab from a Human Monoclonal Antibody Against gp 41 of Human Immunodeficiency Virus Type 1 Elena Casale’, Elisabeth Wenisch’, Xiao-min He’, Pier Giorgio Righetti3 Robert S. Snyder’, Alois Jungbauer’, Christa Tauer*, Florian Ruke? and Daniel C. Carter’? ‘The Space Science Laboratory, Marshall Space Flight Center Huntsville, AL 35812, U.S.A. 2The Institute for Applied Microbiology University of Agriculture and Forestry Peter-Jordan-Strasse 82, A-1190 Vienna, Austria 3Department of Biomedical Sciences and Technologies University of Milano, Via Celoria 2, Milan, Italy 20133 (Received 10 August
1990; accepted 13 August
1990)
A monoclonal IgG antibody directed against gp 41 from the human immunodeficiency virus (HIV-l) 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 P2,2,21 with unit cell constants of a = 665 A, b = 74.3 a and c = 105.3 8. There is one molecule of Fab in the asymmetric unit. The Fab crystals show diffraction to d-spacings less than 3.0 A.
transmembrane protein gp 41, which are suitable for the determination of the three-dimensional structure. The antibody was produced from a xeno hybridoma line 3D6 (Grunow et al., 1988) as part of a research effort involving the large-scale production of monoclonal antibodies to HIV-l (Jungbauer et al., 1989). The isolation and purification of the antibody isoforms produced by this method has been extensively studied (Wenisch et al., 1989; Righetti et al., 1989, 1990). This antibody corresponds to the subclass IgG, and exhibits antibodydependent cellular cytotoxicity. The isolation and purification of the IgG, monoclonal antibody isoprotein was accomplished by utilizing preparative isoelectric focusing in immobilized pH gradients (Wenisch et al., 1989). The Fab fragments were produced by cleavage using papain (Sigma Chemical Company) and purified by f.p.1.c. chromatofocusing and gel filtration methods. The proteolysis was terminated prior to complete digestion of the antibody by monitoring the enzymatic reaction with SDS/polyacrylamide gel electrophoresis. Although this strategy produced a less than optimum yield of Fab, it was done so in an effort to produce a homogeneous Fab preparation. The procedure for producing the Fab fragment is as
The nature of antigen-antibody recognition at the atomic level has been the subject of numerous crystallographic studies and several reviews on the subject have been published (Alzari et al., 1988; Davies et al., 1988; Laver et al., 1990). More recently, progress has been made toward the determination of structures of smaller Fv proteins produced by recombinant methods (Glockshuber et al., 1990: Boulot et al., 1990). Together these approaches should provide for a more complete understanding of antibody-antigen interaction and promise avenues for new therapeutic treatments to a variety of immunologically recognizable illnesses including those of the acquired immunodeficiency syndrome (AIDS). Here, we report crystals of the Fab fragment of a monoclonal antibody to the human immunodeficiency virus -(HIV-l $) directed against an immunodominant epitope of the viral t Author to whom all correspondence should be addressed at: National Aeronautics and Space Administration, ES 76, Biophysics, Huntsville, AL 35812, U.S.A. $ Abbreviations used: HIV-l, human immunodeficiency virus; PEG, polyethylene glycol; f.p.l.c.,
fast protein
liquid
chromatography.
OO22-2836/QO/23051 l-02 $03.00/O
511 c 1990 Academic Press Limited
E. Casale et al.
512
follows: antibody (20 mg) was dissolved in 10 ml of standard PBS buffer containing 3 mM-EDTA, 10 mM-cysteine. Mercury papain was dissolved in the same buffer (0.1 mg/ml) and incubated for 15 minutes to activate the enzyme. The papain and antibody were combined (1 : 400) and incubated for 1.5 hours at 37°C. The reaction was quenched with a solution of iodoacetamide and left in the dark at 4°C for 30 minutes. The Fab was purified by the method of ehromatofocusing on a Pharmacia f.p.1.c. Mono P column using a pH gradient from 9.0 to 6.0 (starting buffer: 0075 M-Tris-acetic acid (pH 9.3); eluting buffer: Pharmacia polybuffer PB96 (pH 6.0)). The Fab was separated from the polybuffer by gel filtration chromatography using a Pharmacia f.p.1.c. Superose 12 column (O.O5w-sodium phosphate (pH 7.0)), then dialyzed against distilled water and lyophilized prior to beginning the crystallization experiments. Cryst’als of Fab were grown by the standard hangmg-drop vapor-diffusion method using solutions of 25 to 30% (w/v) polyethylene glycol (PEG) 3350 (Sigma) and @05 M-sodium phosphate buffer (between pH 7.0 and 7.5) at 22°C. Protein solutions cont’aining 5 ~1 (I 0 mg/ml) of Fab were mixed with 5 ~1 of 20 to 350/I PEG 3350, placed on glass cover slips and inverted and sealed over reservoirs containing the same PEG solution. Plate-like crystals appeared after 3 days and reached ti5 mm x @5 mm x @I mm in size after ten days to three weeks. Crystals of the intact antibody were grown by the method described above except with solutions
of 25 to 350/b (v/v)
2,4-methylpentanediol.
protein concentration of 20 mg/ml, and pH of 8.0 (005 M-sodium phosphate). These crystals grow as tetragonal-like prisms with dimensions too small for conventional X-ray analysis ( the data fall off rapidly Edited
at
higher resolution. Based on an average partial specific volume of 0.74 cm3,lg for proteins and an Mr for the Fab of approximately 47,500, the Matthews coefficient Vm (Matthews, 1968) is 2.74. This implies a crystal solvent content of 55% and the presence of one molecule in the asymmetric unit. Efforts to form crystals containing antigenantibody complexes and solve the crystal structure by
the
method
of molecular
replacement
are
in
progress. This research was supported
Spare
in part by the Office of
and Applications of the 1Jational and Space Administ~ration (NASA) (RTOP
Science
Aeronautics 674-23-OS-17), the Italian Space Agency (AST) and thtl Federal Ministry of Science and Research of the Austrian Government (project no. 7/62). E.C. and X.-M.H. werr supported by NASA under contract, with the lyniversities Space Research Association (ITSRA). ,I. Herrorr is thanked for helpful discussions and for sharing his many crystallization experiences with Fah fragments.
References Alzari.
1’. M., Lasrombr,
Annw.
Rev. Immunol.
M. 13. & Poljack,
Ii. J. (1988).
6, 555-580.
Boulot, G.; ECisele,
Crystallization of the Fab from a Human Monoclonal Antibody Against gp 41 of Human Immunodeficiency Virus Type 1 Elena Casale’, Elisabeth Wenisch’, Xiao-min He’, Pier Giorgio Righetti3 Robert S. Snyder’, Alois Jungbauer’, Christa Tauer*, Florian Ruke? and Daniel C. Carter’? ‘The Space Science Laboratory, Marshall Space Flight Center Huntsville, AL 35812, U.S.A. 2The Institute for Applied Microbiology University of Agriculture and Forestry Peter-Jordan-Strasse 82, A-1190 Vienna, Austria 3Department of Biomedical Sciences and Technologies University of Milano, Via Celoria 2, Milan, Italy 20133 (Received 10 August
1990; accepted 13 August
1990)
A monoclonal IgG antibody directed against gp 41 from the human immunodeficiency virus (HIV-l) 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 P2,2,21 with unit cell constants of a = 665 A, b = 74.3 a and c = 105.3 8. There is one molecule of Fab in the asymmetric unit. The Fab crystals show diffraction to d-spacings less than 3.0 A.
transmembrane protein gp 41, which are suitable for the determination of the three-dimensional structure. The antibody was produced from a xeno hybridoma line 3D6 (Grunow et al., 1988) as part of a research effort involving the large-scale production of monoclonal antibodies to HIV-l (Jungbauer et al., 1989). The isolation and purification of the antibody isoforms produced by this method has been extensively studied (Wenisch et al., 1989; Righetti et al., 1989, 1990). This antibody corresponds to the subclass IgG, and exhibits antibodydependent cellular cytotoxicity. The isolation and purification of the IgG, monoclonal antibody isoprotein was accomplished by utilizing preparative isoelectric focusing in immobilized pH gradients (Wenisch et al., 1989). The Fab fragments were produced by cleavage using papain (Sigma Chemical Company) and purified by f.p.1.c. chromatofocusing and gel filtration methods. The proteolysis was terminated prior to complete digestion of the antibody by monitoring the enzymatic reaction with SDS/polyacrylamide gel electrophoresis. Although this strategy produced a less than optimum yield of Fab, it was done so in an effort to produce a homogeneous Fab preparation. The procedure for producing the Fab fragment is as
The nature of antigen-antibody recognition at the atomic level has been the subject of numerous crystallographic studies and several reviews on the subject have been published (Alzari et al., 1988; Davies et al., 1988; Laver et al., 1990). More recently, progress has been made toward the determination of structures of smaller Fv proteins produced by recombinant methods (Glockshuber et al., 1990: Boulot et al., 1990). Together these approaches should provide for a more complete understanding of antibody-antigen interaction and promise avenues for new therapeutic treatments to a variety of immunologically recognizable illnesses including those of the acquired immunodeficiency syndrome (AIDS). Here, we report crystals of the Fab fragment of a monoclonal antibody to the human immunodeficiency virus -(HIV-l $) directed against an immunodominant epitope of the viral t Author to whom all correspondence should be addressed at: National Aeronautics and Space Administration, ES 76, Biophysics, Huntsville, AL 35812, U.S.A. $ Abbreviations used: HIV-l, human immunodeficiency virus; PEG, polyethylene glycol; f.p.l.c.,
fast protein
liquid
chromatography.
OO22-2836/QO/23051 l-02 $03.00/O
511 c 1990 Academic Press Limited
E. Casale et al.
512
follows: antibody (20 mg) was dissolved in 10 ml of standard PBS buffer containing 3 mM-EDTA, 10 mM-cysteine. Mercury papain was dissolved in the same buffer (0.1 mg/ml) and incubated for 15 minutes to activate the enzyme. The papain and antibody were combined (1 : 400) and incubated for 1.5 hours at 37°C. The reaction was quenched with a solution of iodoacetamide and left in the dark at 4°C for 30 minutes. The Fab was purified by the method of ehromatofocusing on a Pharmacia f.p.1.c. Mono P column using a pH gradient from 9.0 to 6.0 (starting buffer: 0075 M-Tris-acetic acid (pH 9.3); eluting buffer: Pharmacia polybuffer PB96 (pH 6.0)). The Fab was separated from the polybuffer by gel filtration chromatography using a Pharmacia f.p.1.c. Superose 12 column (O.O5w-sodium phosphate (pH 7.0)), then dialyzed against distilled water and lyophilized prior to beginning the crystallization experiments. Cryst’als of Fab were grown by the standard hangmg-drop vapor-diffusion method using solutions of 25 to 30% (w/v) polyethylene glycol (PEG) 3350 (Sigma) and @05 M-sodium phosphate buffer (between pH 7.0 and 7.5) at 22°C. Protein solutions cont’aining 5 ~1 (I 0 mg/ml) of Fab were mixed with 5 ~1 of 20 to 350/I PEG 3350, placed on glass cover slips and inverted and sealed over reservoirs containing the same PEG solution. Plate-like crystals appeared after 3 days and reached ti5 mm x @5 mm x @I mm in size after ten days to three weeks. Crystals of the intact antibody were grown by the method described above except with solutions
of 25 to 350/b (v/v)
2,4-methylpentanediol.
protein concentration of 20 mg/ml, and pH of 8.0 (005 M-sodium phosphate). These crystals grow as tetragonal-like prisms with dimensions too small for conventional X-ray analysis ( the data fall off rapidly Edited
at
higher resolution. Based on an average partial specific volume of 0.74 cm3,lg for proteins and an Mr for the Fab of approximately 47,500, the Matthews coefficient Vm (Matthews, 1968) is 2.74. This implies a crystal solvent content of 55% and the presence of one molecule in the asymmetric unit. Efforts to form crystals containing antigenantibody complexes and solve the crystal structure by
the
method
of molecular
replacement
are
in
progress. This research was supported
Spare
in part by the Office of
and Applications of the 1Jational and Space Administ~ration (NASA) (RTOP
Science
Aeronautics 674-23-OS-17), the Italian Space Agency (AST) and thtl Federal Ministry of Science and Research of the Austrian Government (project no. 7/62). E.C. and X.-M.H. werr supported by NASA under contract, with the lyniversities Space Research Association (ITSRA). ,I. Herrorr is thanked for helpful discussions and for sharing his many crystallization experiences with Fah fragments.
References Alzari.
1’. M., Lasrombr,
Annw.
Rev. Immunol.
M. 13. & Poljack,
Ii. J. (1988).
6, 555-580.
Boulot, G.; ECisele,
