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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Keywords: affinity binding/monoclonal antibody/point mutation/protein A
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Introduction |
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Materials and methods |
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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 T252MT254S, 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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Expression and purification of fusion proteins
CHO-K1 cells were cultured in Dulbeccos 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 manufacturers 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 TrisHCl (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 TrisHCl, 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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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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References |
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Received December 3, 2002; revised February 17, 2003; accepted February 26, 2003.