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Protein Engineering vol.14 no.10 pp.803–806, 2001 Anti-c-myc antibody 9E10: epitope key positions and variability characterized using peptide spot synthesis on cellulose K.Hilpert1, G.Hansen1, H.Wessner1, G.Küttner1, K.Welfle2, M.Seifert3 and W.Höhne4 1Institut für Biochemie, Universitätsklinikum Charité, Humboldt-Universität zu Berlin, Monbijoustr. 2, 10117 Berlin, 2Max-Delbrück-Centrum für Molekulare Medizin, Robert-Rössle-Straβe 10, 13122 Berlin, 3Institut für Medizinische Immunologie, Universitätsklinikum Charité, Humboldt-Universität zu Berlin, Monbijoustr. 2, 10117 Berlin, Germany 4To whom correspondence should be addressed. E-mail: [email protected] The 9E10 antibody epitope (EQKLISEEDL) derives from a protein sequence in the human proto-oncogen p62c-myc and is widely used as a protein fusion tag. This myc-tag is a powerful tool in protein localization, immunochemistry, ELISA or protein purification. Here, we characterize the myc-tag epitope by substitutional analysis and length variation using peptide spot synthesis on cellulose. The key amino acids of this interaction are the core residues LISE. The shortest peptide with a strong binding signal is KLISEEDL. Dissociation constants of selected peptide variants to the antibody 9E10 were determined. scFv constructs with the shortest possible myc-tags were successfully detected by Western blot and ELISA, giving a signal comparable to that of the original myc-tag. Keywords: epitope characterization/MAB 9E10/myc-tag/ peptide spot synthesis/substitutional analysis Introduction The monoclonal antibody 9E10, obtained by immunizing a BALB/c mouse with a peptide from the C-terminal region (amino acid 408–439) of the human c-myc protein (Evan et al., 1985), is an immunoglobulin of the subclass IgG1/κ that binds specifically to human c-myc. The c-myc gene product, a 62 kDa proto-oncoprotein (p62c-myc), is mainly localized in the nucleus of the cell and its expression levels are elevated in a variety of hematopoietic tumors. Therefore, p62 is extensively used in cancer research and it has been proven to be a diagnostic parameter in various types of cancer (Fuchs et al., 1997). The 9E10 epitope has also become a well-known affinity tag used in recombinant protein expression systems such as bacteria, yeast or insect cells. Together, this myc-tag and the 9E10 antibody can be used to detect recombinant proteins by immuno-staining of blots or in ELISAs, or to determine the localization of a protein in a cell. In addition, a protein labeled with the myc-tag can be purified in a single-step procedure using a 9E10 antibody-coupled affinity column (Kramer et al., 1997). Numerous publications describe the successful application of this protein fusion tag (Hoogenboom et al., 1991; Dubel et al., 1993, 1995; Kleymann et al., 1995; Kontermann et al., 1995; Manstein et al., 1995; TerBush and Novick, 1995; Bach et al., 1996; Tanaka et al., 1996; Schiweck et al., 1997). The myc-tag amino acid sequences commonly found in practical use are: EQKLISEEDLN (Hoogenboom et al., 1991) and EEQKLISEEDL (Fuchs et al., 1997). The Fv © Oxford University Press fragment of the monoclonal antibody 9E10 has been sequenced and also cloned and expressed in Fv, Fab and scFv formats (Fuchs et al., 1997; Schiweck et al., 1997). Materials and methods Peptide synthesis Cellulose-bound peptides for binding experiments were prepared using a pipetting robot (Abimed, Langenfeld, Germany) or by pipetting by hand using Whatman 50 cellulose membranes (Whatman, Maidstone, UK), as described previously (Kramer et al., 1994; Kramer and Schneider-Mergener, 1998). Deviating from these protocols, the protection group cleavage procedure was changed as follows: incubation of the membrane for 3 h in 90% TFA (Fluka, Deisenhofen, Germany), 2% triisopropylsilan (Lancaster Synthesis GmbH, Mühlheim am Main, Germany), 5% water and 3% phenol (Fluka), change of the solution after 1 h without washing, shaking gently. The peptides are coupled to the cellulose support by their C-terminus with two β-alanine as a spacer in between. The peptides used for Kd determination were prepared by Interactiva (Ulm, Germany), or in the laboratory of J.Schneider-Mergener according to standard Fmoc machine protocols using a multiple peptide synthesizer (Abimed). Binding experiments with cellulose-bound peptides The cellulose membranes were washed with 96% ethanol for 5 min and equilibrated with Tris-buffered saline (20 mM Tris– HCl and 0.5 M NaCl, pH 7.5) containing 0.01% Tween 20 (TTBS). The membranes were blocked by incubation with blocking agent (Genosys, Cambridge, UK) in 0.1 M Tris buffer, pH 7.5, for 2 h at room temperature, washed three times with TTBS for 3 min each. After that the membrane was incubated in a solution containing 2 µg/ml 9E10, a sheep anti-mouse IgG horseradish peroxidase (HRP) conjugate (Amersham, Freiburg, Germany) (dilution 1:500) and 3% Gelifundol (Biotest Pharma GmbH, Dreieich, Germany) in TTBS for 2 h at room temperature or overnight at 4°C. The membranes were finally washed with TTBS three times for 10 min and then incubated with a chemiluminescence substrate (Pierce, IL, USA) for 3 min or incubated in diaminobenzidine solution. The light arising from spots with bound 9E10 antibody (in the case of the chemiluminescence reaction) was measured with a chemiluminescence detector (Boehringer, Mannheim, Germany). A diaminobenzidine solution containing 3,3⬘-diaminobenzidine tetrahydrochloride (0.8 mg/ml) (Sigma, Deisenhofen, Germany) in 0.1 M Tris–HCl, pH 7.5, with 0.2% NiCl2 and 0.03% H2O2 was used to detect peroxidase activity (in the case of Figure 1a). The 9E10 antibody was produced and purified from hybridoma cell line 9E10 in our group. scFv fragment construction, recombinant expression and purification The single chain antibody fragment 1F9 specifically binding to turkey egg white lysozyme (Küttner et al., 1998; Ay et al., 803 K.Hilpert et al. Fig. 1. Substitutional matrix of (a) the original and (b) a shortened myc-tag peptide. The first column on the left represents peptides with the wild-type sequence: EQKLISEEDLN (a) and KLISEEDL (b). The lines represent peptides substituted at that position by 19 coded amino acids. The bound antibody 9E10 was detected by HRP-labeled anti-mouse IgG and incubation in a diaminobenzidin solution (a) or with a chemiluminescence substrate (b). The light signal from (b) is shown as a negative image, the strongest binding resulting in the darkest spot. 2000) was used as a model protein. Base sequences coding for two shortened myc-tags (LISEEDL and LISEFEL) containing an EcoRI restriction site at the C-terminus were introduced by PCR. These tags were fused to the C-terminus (framework IV—KLEIK) of the scFv. The gene for the antibody fragment was recloned then into pHEN 1 (Hoogenboom et al., 1991) using SfiI and EcoRI for digestion. The mutant constructs were transformed into Escherichia coli TG 1 cells and screened for expression of the scFv product. The correct nucleotide sequence of all constructs was confirmed by sequencing using the dideoxy method (Sanger et al., 1977). The cells were grown overnight in 2⫻ TY medium containing ampicillin and 1% glucose for catabolite repression. After centrifugation of the cells the pellet was suspended in fresh medium containing ampicillin and 0.1 mM isopropyl β-D-thiogalactopyranoside (IPTG). The expression was continued for 4 h at 25°C. After cell lysis the screening of total lysates was carried out by Western blotting using the anti-myc-tag 9E10 antibody and an anti-mouse HRP conjugate as the secondary antibody. 3,3⬘,5,5⬘Tetramethylbenzidine blue (TMB) (Seramun GmbH, Dolgenbrodt, Germany) was used for the color development. The clones showing the highest expression for both of the mutants were used for scFv production. The cells were cultured and induced as described above and the periplasmic fraction was isolated by incubation of the cell pellet with 1/100 of the culture volume using 0.1 M Tris–HCl pH 8.0, 0.5 M saccharose, 1 mM ethylenediamintetraacetic acid (EDTA). The periplasmic fractions were dialyzed against 50 mM Tris–HCl pH 8.0, 0.15 M NaCl and bound to a BrCN Sepharose 6 column containing coupled turkey egg white lysozyme. After washing with 0.2 M glycine, 0.2 M NaCl pH 5.0, the bound antibody fragment was eluted by 0.2 M glycine, 0.2 M NaCl pH 2.0. The pooled fractions were dialyzed against phosphate buffered saline and concentrated using Microcon 10 columns (Millipore GmbH, Eschborn, Germany). The purified proteins 804 were diluted to the same concentration each (concentration was measured using absorption at 280 nm) and the band intensity of the Western blots was visually compared. The Western blot analysis was carried out using 17% SDS polyacrylamide gels and blotting onto polyvinylidene fluoride (PVDF) membranes. After blocking the membrane for 1 h with 5% dry milk in TTBS and three times washing with TTBS the membrane was incubated with 1 µg/ml 9E10 antibody and anti-mouse Fcgamma antibody-HRP conjugate 1:2000 dilution (Sigma GmbH, Taufkirchen, Germany) simultaneously in TTBS containing 5% gelatine for 1.5 h at room temperature and washed three times with TTBS. For the color development TMB blue was used. Kd measurements with peptides The microtiter plates were coated with the anti-pre-S2 (HBV) scFv 1F6 containing the fused myc-tag sequence EQKLISEEDLN (Kuttner et al., 1999) (50 µl per well at 1.5 µg/ml) overnight at 4°C with a 0.1 M carbonate buffer pH 9.6. The binding of the 9E10 antibody (0.25 µg/ml) to this scFv was competed by different concentrations of the peptides. The bound antibody was detected by a sheep anti-mouse IgGHRP conjugate (Amersham) (dilution 1:1000). The Kd was determined with the method described by Friguet (Friguet et al., 1985). Results and discussion The myc-tag sequence EQKLISEEDLN included in vectors such as the pHEN antibody phage display vector (Hoogenboom et al., 1991) was used for epitope characterization of the 9E10 antibody. The C-terminal asparagine in this tag sequence, which is a leucine at this position in the c-myc protein, is a product of vector construction (Munro and Pelham, 1986). To investigate the myc-tag/9E10 interaction we carried out a substitutional analysis of the sequence EQKLISEEDLN using the method of peptide spot synthesis on cellulose sheets (Frank, 1992; Kramer et al., 1994). Each amino acid of the peptide was substituted by the other 19 coded amino acids to evaluate the importance of each position of the sequence. The cellulose sheet was incubated in 9E10 solution and after a washing procedure the bound antibody was detected by a HRP-labeled anti-mouse antibody (see Figure 1a). Peptide positions where amino acids can only be exchanged by a reduced set of, mainly physico-chemically similar, amino acids are referred to as key positions; the results show that these are localized in the core of the sequence. Leucine at position 4 can only be substituted by itself, whereas isoleucine (position 5) can also be replaced by physico-chemically similar amino acids, and serine (position 6) by glycine, proline and alanine. Furthermore, glutamic acid at position 7 can be changed to glutamine and also to several hydrophobic amino acids (phenylalanine, tryptophan, methionine, tyrosine) but not to aspartic acid. Interestingly, the amino acids at positions 8 and 9 do not show interaction specificity, whereas leucine in the next position (10) is again more specific. The introduction of proline in any one of the positions from 3 to 10, with the exception of position 6, is unfavorable for this interaction. The substitution of serine at position 6 by proline or glycine may indicate that the peptide adopts a turn conformation at the antibody binding site. A next step involved a truncation of the antibody epitope by altering the length of the sequence EQKLISEEDLN synthesized on cellulose. The sequence was shortened C- or Epitope variability of the anti-c-myc antibody 9E10 Table I. Affinity determination for peptides in solution Peptide sequence Kd⫻107 (M) (ELISA) EQKLISEEDLN KLISEEDL FLISEEDL QLISEEDL KLISDEDL KLISEFEL QKLISEFELN EQKLISEFELN TMFLISEEDLQ 5.6 ⫾ 260 ⫾ 200 ⫾ 820 ⫾ ⬎2000 73 ⫾ 14 ⫾ 1.9 ⫾ 12 ⫾ 1.9 190 110 430 Kd values were measured by competition ELISA: for the original myc peptide EQKLISEEDLN; a peptide with randomly chosen amino acids at position 1, 2, and 11, and a change at position 3 from lysine to phenylalanine; and different length variants of the core sequence of the mutated myc-tag KLISEFEL. Additional peptide variants are included from substitutional analysis of KLISEEDL. Fig. 2. Western blot of purified scFv 1F9 variants with myc-tag fusions. Shortened (snmyc) and shortened and mutated (mutmyc) myc-tags are compared to sc-Fv containing the original myc-tag sequence (EQKLISEEDLN). The scFv variants with the changed myc-tags have as short a tag as possible, the last three amino acids from the light chain serving as the first amino acids of the tag. The scFv with the original myctag has a spacer between the light chain and the myc sequence comprising three alanine residues resulting from the vector construction. The scFvs were purified and used at the following concentrations (determined by absorption at 280 nm): 1 ⫽ 140, 2 ⫽ 70, 3 ⫽ 35, 4 ⫽ 17.5 and 5 ⫽ 8.75 µg/ml. The scFv fusion proteins were detected by a combination of 9E10/anti-mouse Fcgamma HRP-labeled antibodies and visualized by the color reaction of a HRP substrate. N-terminally or at both ends (data not shown). Whereas the binding signal decreases in the case of the peptide without asparagine at position 11, the N-terminally shortened peptides both without glutamic acid of position 1 and glutamine of position 2 lead to a binding signal comparable to that of the untruncated peptide. A drastic effect amounting to the complete loss of detectable binding signal is associated with further shortening by omitting the lysine at position 3. The shortest peptide giving a strong binding signal, although weaker than the complete 11-mer myc-tag sequence, is the 8-mer peptide KLISEEDL. Additionally, we synthesized a substitutional analysis for the truncated peptide to compare with the wildtype substitutional analysis (Figure 1b). The results show that the key amino acids remain the same as in the wild-type myctag. Lysine at position 3, glutamic acid at position 7 and leucine at position 10 (numbering according to the initial 11-mer peptide) appear to become more specific, since fewer substitutions are tolerated. However, this is probably due to the overall reduced binding affinity of the 8-mer (Table I), thus diminishing the saturation effect and revealing intrinsic differences between the signals. Therefore, some peptide variants from the substitutional analysis of the shortened peptide KLISEEDL were selected to measure the Kd values in solution by competition ELISA: FLISEEDL with a binding signal similar to the wild-type, QLISEEDL with a somewhat lower binding signal and KLISEDEL with no binding signal at all. The Kd values obtained (Table I) reflect the results from the binding studies of substitutional analysis very well. The substitutional analysis of the shortened peptide KLISEEDL was inspected for variants with higher affinity. Interestingly, stronger binding signals resulted from substituting glutamic acid at position 8 by phenylalanine and aspartate at position 9 by glutamate (Figure 1b). Substitutional analysis of a new peptide with the glutamate at position 8 exchanged for phenylalanine, KLISEFDL, again showed a slightly higher binding signal at position 9 for the substitution of aspartic acid to glutamic acid (data not shown). For the peptide sequence exchanged both in positions 8 and 9, KLISEFEL, the substitutional analysis did no more show any significantly stronger binding signal (data not shown). Several peptides with different lengths based on the optimized core sequence KLISEFEL were now synthesized and the affinities of the soluble peptides measured by competition ELISA (see Table I). The 8-mer peptide KLISEFEL showed higher affinity than KLISEEDL, but, as expected, decreased affinity to 9E10 compared to the original myc-tag EQKLISEEDLN, as did the 10-mer QKLISEFELN. However, the 11-mer EQKLISEFELN bound to the antibody somewhat better than the original myc-tag. In addition, a peptide with randomly chosen amino acids at positions 1, 2, and 11 and, based on the substitution analysis results of the shortened myc sequences, a change at position 3 from lysine to phenylalanine, (TMFLISEEDLQ) was synthesized and its affinity to the 9E10 antibody was also determined (Table I). The affinity decreased only by a factor of approximately 2 compared to the original myc-tag. The results demonstrate that it is possible to substitute positions 1, 2, 11 and with respect to the data from Figure 1b also position 3. The Kd determination showed that the original myc peptide sequence EQKLISEEDLN has a 7-fold lower affinity to the antibody 9E10 compared to the fluorescent Abz-EQKLISEEDLN peptide which was determined by fluorescence titration to a Kd ⫽ 8 ⫾ 0.5⫻10–8 M (Schiweck et al., 1997). Because of that deviation we performed an independent affinity determination by isothermal titration calorimetry for the peptide EQKLISEEDLN, resulting in a Kd of 5.3 ⫾ 0.3⫻10–7 M. This is very close to the value obtained from the ELISA experiment. The deviation between the fluorescence titration result and our data may be due to an additional interaction between the 2-aminobenzoyl group and the antibody combining site thus contributing to affinity. To look for specific interactions of additional amino acids of a C- and N-terminally elongated myc-tag, a substitutional array of the peptide AEEQKLISEFELLRK was synthesized and analyzed (data not shown), but no further key amino acids or substitutionally sensitive positions were detected. Taking into account the information accumulated so far we designed single chain Fv fragments with a VH/VL orientation where the last three amino acids of the scFv framework correspond to the first three amino acids of the myc-tag. The resulting tags were EIKLISEEDL, and the mutated myc-tag EIKLISEFEL, where EIK derive from the light chain variable region of the scFv and the N-terminal asparagine was omitted. This means that the myc-tag of the recombinant protein is kept as short as possible, thus minimizing antigenicity effects and the influence of these residues on the protein itself. This can be an advantage for example in protein crystallization, measurements of intracellular protein function or activity, or 5.6 2.1 0.36 1.4 805 K.Hilpert et al. in the case of recombinant proteins for therapeutical use. Western blot analyses showed that based on the results of the substitutional analysis it is possible to shorten and mutate the original myc-tag without loss of sensitivity as determined by a dilution series with the scFv variants of the 9E10 antibody (Figure 2). A possible cross-reactivity between the murine scFv and anti-mouse-HRP secondary antibody used for 9E10 detection was avoided by use of an anti-mouse Fcgamma-specific secondary antibody. The sensitivity of the different myc-tags against proteolyses was analyzed by a 6-day incubation of the purified scFvs mixed with a cytoplasmic extract of E.coli TG1 cells at 37°C. No proteolytic cleavage of the three myc-tags was seen over this time period as confirmed by Western blotting (data not shown). Our results show that is possible to modify and optimize the commonly used myc-tag for specific applications using information gained through substitutional analysis. For example, based on our results Paschke et al. (Paschke et al., 2001) developed a mutated myc-tag that contains a protease cleavage site. Other options include substitutions leading to reduced affinity, which may be an advantage in protein affinity purification where more gentle elution conditions can then be applied. 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