Protein Engineering, Vol. 16, No. 4, 243-245,
April 2003
© 2003 Oxford University Press
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Single amino acid substitution in the mouse IgG1 Fc region induces drastic enhancement of the affinity to protein A
Department of Biomolecular Engineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan
1 To whom correspondence should be addressed.E-mail: takaike{at}bio.titech.ac.jp
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Abstract |
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The purification of monoclonal antibody sometimes requires a lot of time and involves complicated steps because of the poorer ability of mouse IgG to interact with protein A, or also with protein G, than IgGs from other species such as those of human and rabbit. To resolve this problem, we exchanged one or two amino acid residues of mouse IgG Fc region with that of human IgG. Three mutants (T252M, T254S and T252M–T254S) showed significant improvement in the affinity to protein A. The exchange of the threonine 252 residue to methionine (T252M) was most efficient. This result suggests that a direct and simple modification allows the efficient purification of monoclonal antibody and of fusion protein containing mouse IgG Fc region.
Keywords: affinity binding/monoclonal antibody/point mutation/protein A
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Recently, the production and application of monoclonal antibody has been considered a general and useful method in both biomedical technology and biology. Modification and construction of fusion protein designed by the mouse IgG Fc region and other functional proteins also have great potential for application to the observation of many biological phenomena (Monfardini et al., 1998
; Adachi et al., 2002; Huynh-Do et al., 2002). There are, however, several problems with the purification of these antibodies and fusion proteins. The main reason is the low affinity of mouse immunoglobulin G (IgG), especially IgG1, to protein A from Staphylococcus aureus, which can bind to the Fc portion of IgG. IgG1 is the major subclass produced by hybridomas. To improve the affinity of mouse IgG1 to protein A-conjugated gel beads, increasing the salt concentration and adjusting the pH to the alkaline range were effective. Under these conditions, however, non-specific interactions between protein A and other proteins, especially serum proteins, could not be neglected. Because of the low affinity of mouse IgG1, the Fc region of human IgG or other species was usually used for the construction of fusion proteins to improve the affinities of Fc residues. When these fusion proteins and also humanized antibodies were used for in vivo studies, several problems arose, e.g. rejection of injected molecules by immune response and induction of unexpected inflammations. In this study, we showed that the replacement of only one or two amino acid residues of the mouse IgG1 Fc domain drastically enhanced the affinity to protein A. |
Materials and methods |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Site-directed mutagenesis
Fusion protein of mouse E-cadherin extracellular domain and mouse IgG1 Fc domain was used as a target of mutagenesis (Nagaoka et al., 2002). To construct mutants, plasmid vector pGEM-Fc containing mouse IgG1 Fc domain cDNA was used as a PCR template.
To induce mutations, the following oligonucleotide pairs (ESPEC Oligo Service, Tsukuba, Japan) were used (Figure 1B): for T252M, 5'-GGA TGT GCT CAT GAT TAC TCT GAC TCC-3' and 5'-GGA GTC AGA GTA ATC ATG AGC ACA TCC-3'; for T254S, 5'-GGA TGT GCT CAC CAT TTC TCT GAC TCC-3' and 5'-GGA GTC AGA GAA ATG GTG AGC ACA TCC-3'; and for double mutation T252M–T254S, 5'-GGA TGT GCT CAT GAT TTC TCT GAC TCC-3' and 5'-GGA GTC AGA GAA ATC ATG AGC ACA TCC-3', where underlines indicate the replaced nucleotides. Site-directed mutagenesis was generated by performing PCR using Pfu TurboTM DNA polymerase (Stratagene Cloning Systems, La Jolla, CA) followed by digestion of template DNA with DpnI (Invitrogen, Carlsbad, CA). Reaction mixtures were used directly for transformation and mutants were verified with the Gene Rapid DNA sequencer (Amersham Biosciences, Piscataway, NJ).
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To construct the expression vector for mutated fusion protein, pGEM-EC containing mouse E-cadherin extracellular domain cDNA and mutated pGEM-Fc were digested with HindIII–NotI or NotI–XbaI, respectively, and ligated with pRC/CMV (Invitrogen) fragment digested with HinIII and XbaI.
Expression and purification of fusion proteins
CHO-K1 cells were cultured in Dulbecco’s modified Eagle medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum (FBS) at 37°C in 5% CO2. Expression vectors were introduced into CHO-K1 cells with lipofectamine reagent (Invitrogen) according to manufacturer’s directions. After the cells had been cultured for 2 days in medium supplemented with 400 µg/ml G418 (Invitrogen), conditioned media were collected and centrifuged to remove the cells and debris. The fusion proteins were purified from conditioned media using an rProtein A FF column (Amersham Biosciences). After loading the sample, the column was washed with 20 mM phosphate buffer (pH 7.0). Fractions eluted with 0.1 M sodium citrate (pH 2.7) were neutralized by adding a one-fifth volume of 1 M Tris–HCl (pH 9.0). All fractions (flow-through, wash and eluate) were collected and analyzed by western blotting.
Western blotting analysis
Collected fractions were diluted with PBS to adjust the volume to 5 ml and denatured by boiling with an equal volume of Laemmli sample buffer (100 mM Tris–HCl, pH 6.8 containing 4% SDS, 12% 2-mercaptoethanol and 20% glycerol) for 5 min. Samples were separated by 7.5% polyacrylamide gel electrophoresis and transferred electrophoretically to a poly-(vinylidene difluoride) (PVDF) membrane (Immobilon-P; Millipore, Bedford, MA). Mutated and wild-type Fc fragment-containing E-cadherin fusion protein was detected with horseradish peroxidase (HRP)-conjugated anti-mouse antibody followed by ECL reagent (Amersham Biosciences).
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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The amino acid sequence of immunoglobulin G heavy chain keeps a high homology regardless of the species (Figure 1A
). There are, however, several differences, including the important residues for the interaction with the B domain of protein A, which mainly interacts with IgG (Deisenhofer et al., 1978; Deisenhofer, 1981; Sauer-Eriksson et al., 1995). Therefore, we examined the effect of exchanging the residues of mouse IgG1 with those of human IgG1. In this study, we focused on two amino acid residues, threonine 252 and threonine 254. In human IgG1, these residues are methionine and serine, respectively. In order to exchange these two residues, PCR was performed using three pairs of oligonucleotides (Figure 1B). After sequencing, DNA fragments containing wild-type or mutated Fc domain were inserted to eukaryotic expression vector containing E-cadherin extracellular domain cDNA. Wild-type and mutated proteins were expressed in CHO-K1 cells.
Conditioned media from transfected cells were collected and purified using an rProtein A FF column. The affinity of the mutated Fc portion to protein A was analyzed by western blotting (Figure 2) or ELISA (data not shown). In both cases, higher affinity was observed when threonine 252 was exchanged to methionine (T252M). Mutations are denoted by the amino acid residue and number, followed by the replaced amino acid. Double mutation is represented by the combination of the single mutations linked by a dash. The mutation of T254S gave a minor improvement in affinity but was less effective than T252M. The improvement in affinity by these mutations seemed to be mainly based on the reduction of steric hindrance and on the change in electrostatic interaction between the Fc region and the B domain of protein A. Since the residues near the interactive core in protein A are highly hydrophobic (Deisenhofer, 1981; Sauer-Eriksson et al., 1995), threonine might be destabilizing the interaction because threonine is more hydrophilic than methionine. The threonine residue of wild-type mouse IgG1 has greater steric hindrance than serine residues. Although the effect of exchanging threonine 254 to serine was unclear, it seems to be because the serine residue is less bulky than threonine. Generally, the affinity of mouse IgG1 to protein A could be improved by increasing the salt concentration to eliminate the water or by raising the pH (>8.0) to deprotonate of the interactive core. These effects derive from increasing the hydrophobicity of Fc domain, which is critical for the interaction. In this study, we confirmed that the point mutation of only one amino acid residue at the CH2 domain could efficiently improve the affinity of mouse IgG1 to protein A. We could suggest that these kinds of point mutation could be applied to the purification of monoclonal antibodies obtained from transgenic mice which have threonine residues replaced by methionine.
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Acknowledgments |
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We are grateful to Professor Motonori Hoshi of Keio University and Ms Maya Kumano of the Tokyo Institute of Technology for their generous gift of hybridoma ku-HD-2A. We thank Dr Maria Carmelita Kasuya of the University of Tokyo for critical reading of the manuscript. This study was supported by a Molecular Synchronization for Design of New Materials Systems Grant-in-Aid for Scientific Research on Priority Area (A) from the Ministry of Education, Science, Sports and Culture of Japan.
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References |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Adachi,O., Nakano,A., Sato,O., Kawamoto,S., Tahara,H., Toyoda,N., Yamato,E., Matsumori,A., Tabayashi,K. and Miyazaki,J. (2002) Gene Ther., 9, 577–583.[CrossRef][Web of Science][Medline]
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Huynh-Do,U., Vindis,C., Liu,H., Cerretti,D.P., McGrew,J.T., Enriquez,M., Chen,J. and Daniel,T.O. (2002) J. Cell Sci., 115, 3073–3081.
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Received December 3, 2002; revised February 17, 2003; accepted February 26, 2003.
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