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• W3111907453 abstract "Proteins are modulated by a variety of posttranslational modifications including methylation. Despite its importance, the majority of protein methylation modifications discovered by mass spectrometric analyses are functionally uncharacterized, partly owing to the difficulty in obtaining reliable methylsite-specific antibodies. To elucidate how functional methylsite-specific antibodies recognize the antigens and lead to the development of a novel method to create such antibodies, we use an immunized library paired with phage display to create rabbit monoclonal antibodies recognizing trimethylated Lys260 of MAP3K2 as a representative substrate. We isolated several methylsite-specific antibodies that contained unique complementarity determining region sequence. We characterized the mode of antigen recognition by each of these antibodies using structural and biophysical analyses, revealing the molecular details, such as binding affinity toward methylated/nonmethylated antigens and structural motif that is responsible for recognition of the methylated lysine residue, by which each antibody recognized the target antigen. In addition, the comparison with the results of Western blotting analysis suggests a critical antigen recognition mode to generate cross-reactivity to protein and peptide antigen of the antibodies. Computational simulations effectively recapitulated our biophysical data, capturing the antibodies of differing affinity and specificity. Our exhaustive characterization provides molecular architectures of functional methylsite-specific antibodies and thus should contribute to the development of a general method to generate functional methylsite-specific antibodies by de novo design. Proteins are modulated by a variety of posttranslational modifications including methylation. Despite its importance, the majority of protein methylation modifications discovered by mass spectrometric analyses are functionally uncharacterized, partly owing to the difficulty in obtaining reliable methylsite-specific antibodies. To elucidate how functional methylsite-specific antibodies recognize the antigens and lead to the development of a novel method to create such antibodies, we use an immunized library paired with phage display to create rabbit monoclonal antibodies recognizing trimethylated Lys260 of MAP3K2 as a representative substrate. We isolated several methylsite-specific antibodies that contained unique complementarity determining region sequence. We characterized the mode of antigen recognition by each of these antibodies using structural and biophysical analyses, revealing the molecular details, such as binding affinity toward methylated/nonmethylated antigens and structural motif that is responsible for recognition of the methylated lysine residue, by which each antibody recognized the target antigen. In addition, the comparison with the results of Western blotting analysis suggests a critical antigen recognition mode to generate cross-reactivity to protein and peptide antigen of the antibodies. Computational simulations effectively recapitulated our biophysical data, capturing the antibodies of differing affinity and specificity. Our exhaustive characterization provides molecular architectures of functional methylsite-specific antibodies and thus should contribute to the development of a general method to generate functional methylsite-specific antibodies by de novo design. Proteins are modified by a variety of posttranslational modifications (PTM) including phosphorylation, acetylation, glycosylation, and methylation, which are known to play key roles in modulating protein function. Protein methylation is one of the most important histone modifications, affecting changes in gene transcription (1Kouzarides T. Chromatin modifications and their function.Cell. 2007; 128: 693-705Abstract Full Text Full Text PDF PubMed Scopus (7315) Google Scholar, 2Jenuwein T. Allis C.D. Translating the histone code.Science. 2001; 293: 1074-1080Crossref PubMed Scopus (7177) Google Scholar). Recently, numerous methylation sites in nonhistone proteins have been discovered, and methylation has been reported to play a role in both fundamental biological processes and disease states such as cancer (3Hamamoto R. Saloura V. Nakamura Y. Critical roles of non-histone protein lysine methylation in human tumorigenesis.Nat. Rev. Cancer. 2015; 15: 110-124Crossref PubMed Scopus (213) Google Scholar, 4Biggar K.K. Li S.S.C. Non-histone protein methylation as a regulator of cellular signalling and function.Nat. Rev. Mol. Cell Biol. 2015; 16: 5-17Crossref PubMed Scopus (248) Google Scholar, 5Lanouette S. Mongeon V. Figeys D. Couture J.F. The functional diversity of protein lysine methylation.Mol. Syst. Biol. 2014; 10: 724Crossref PubMed Scopus (145) Google Scholar). Nearly 1% of human genes encode methyltransferases (6Petrossian T.C. Clarke S.G. Uncovering the human methyltransferasome.Mol. Cell. Proteomics. 2011; 10M110.000976Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar), highlighting the diversity of ligands and the importance of methylation in maintaining homeostasis. Yet the majority of methyltransferases remain to be functionally characterized. Indeed, although growing numbers of methylation sites in nonhistone proteins have been identified using recently developed mass spectrometry–based techniques (7Ong S.E. Mittler G. Mann M. Identifying and quantifying in vivo methylation sites by heavy methyl SILAC.Nat. Methods. 2004; 1: 119-126Crossref PubMed Scopus (345) Google Scholar, 8Geoghegan V. Guo A. Trudgian D. Thomas B. Acuto O. Comprehensive identification of arginine methylation in primary T cells reveals regulatory roles in cell signalling.Nat. Commun. 2015; 6: 6758Crossref PubMed Scopus (82) Google Scholar), the function of specific methylations often remains unknown (4Biggar K.K. Li S.S.C. Non-histone protein methylation as a regulator of cellular signalling and function.Nat. Rev. Mol. Cell Biol. 2015; 16: 5-17Crossref PubMed Scopus (248) Google Scholar, 9Murn J. Shi Y. The winding path of protein methylation research: milestones and new frontiers.Nat. Rev. Mol. Cell Biol. 2017; 8: 517-527Crossref Scopus (72) Google Scholar). Historically, antibodies have played a major role in advancing basic and translational research into PTM. The advent of phosphosite-specific antibodies led to an explosion in phosphorylation research and demonstrated the ubiquity of this protein modification in cellular processes. However, methylsite-specific monoclonal antibodies remain rare to nonexistent (10Hattori T. Lai D. Dementieva I.S. Montaño S.P. Kurosawa K. Zheng Y. Akin L.R. Świst-Rosowska K.M. Grzybowski A.T. Koide A. Krajewski K. Strahl B.D. Kelleher N.L. Ruthenburg A.J. Koide S. Antigen clasping by two antigen-binding sites of an exceptionally specific antibody for histone methylation.Proc. Natl. Acad. Sci. U. S. A. 2016; 113: 2092-2097Crossref PubMed Scopus (19) Google Scholar), and lack of highly specific antibodies often becomes a bottleneck for subsequent functional studies after identifying a new methylation site (11Hattori T. Koide S. Next-generation antibodies for post-translational modifications.Curr. Opin. Struct. Biol. 2018; 51: 141-148Crossref PubMed Scopus (15) Google Scholar). For both ease of use and adaptability to currently available techniques, monoclonal antibodies remain ideal tools for the recognition of site-specific PTM. Although several studies have attempted to create artificial receptors that can recognize methylated lysines (12Gruber T. Synthetic receptors for the recognition and discrimination of post-translationally methylated lysines.ChemBioChem. 2018; 19: 2324-2340Crossref PubMed Scopus (9) Google Scholar), these cannot be easily employed in basic immunochemical assays such as Western blotting, immunocytochemistry, and immunohistochemistry owing to low affinity and a lack of sequence specificity surrounding the modification site. Indeed, protein methylation is considered one of the most difficult targets to create modification-specific antibodies owing to the minute differences in chemical moieties brought by the addition of -CH3 group(s) (10Hattori T. Lai D. Dementieva I.S. Montaño S.P. Kurosawa K. Zheng Y. Akin L.R. Świst-Rosowska K.M. Grzybowski A.T. Koide A. Krajewski K. Strahl B.D. Kelleher N.L. Ruthenburg A.J. Koide S. Antigen clasping by two antigen-binding sites of an exceptionally specific antibody for histone methylation.Proc. Natl. Acad. Sci. U. S. A. 2016; 113: 2092-2097Crossref PubMed Scopus (19) Google Scholar). Selection-based technology (such as phage display) rather than screening-based technologies (such as hybridoma methods) can take advantage of the power of directed evolution to select for antibody clones possessing the desired function, given sufficient selection pressure during iterative rounds of panning. Typically, a peptide sequence containing the target modification is used for immunization, with subsequent selection from the immune repertoire of the immunized animal to obtain the site-specific antibodies. However, the dominant immune response elicits modification-specific antibodies that only recognize peptide antigen in ELISA, while antibodies that also recognize protein antigen in biochemical applications such as Western blot are less common. In particular, most currently available methylation-specific monoclonal antibodies target histone tails, which are unstructured and thought to behave like peptides (1Kouzarides T. Chromatin modifications and their function.Cell. 2007; 128: 693-705Abstract Full Text Full Text PDF PubMed Scopus (7315) Google Scholar, 10Hattori T. Lai D. Dementieva I.S. Montaño S.P. Kurosawa K. Zheng Y. Akin L.R. Świst-Rosowska K.M. Grzybowski A.T. Koide A. Krajewski K. Strahl B.D. Kelleher N.L. Ruthenburg A.J. Koide S. Antigen clasping by two antigen-binding sites of an exceptionally specific antibody for histone methylation.Proc. Natl. Acad. Sci. U. S. A. 2016; 113: 2092-2097Crossref PubMed Scopus (19) Google Scholar). In this study, we generated trimethylated lysine-specific antibodies using a methylated peptide derived from MAP3K2 (Lys260). MAP3K2 is methylated by the oncogenic methyltransferase SMYD3, and methylation of Lys260 has been linked to functional regulation of this kinase (13Colón-Bolea P. Crespo P. Lysine methylation in cancer: SMYD3-MAP3K2 teaches us new lessons in the Ras-ERK pathway.Bioessays. 2014; 36: 1162-1169Crossref PubMed Scopus (26) Google Scholar, 14Mazur P.K. Reynoird N. Khatri P. Jansen P.W.T.C. Wilkinson A.W. Liu S. Barbash O. Van Aller G.S. Huddleston M. Dhanak D. Tummino P.J. Kruger R.G. Garcia B.A. Butte A.J. Vermeulen M. et al.SMYD3 links lysine methylation of MAP3K2 to Ras-driven cancer.Nature. 2014; 510: 283-287Crossref PubMed Scopus (232) Google Scholar). The antibody was created using our original technology that pairs rabbit immunization with selection using fully rabbit (not chimeric) Fab-phage display, and this report can be considered the first detailed characterization of an antibody obtained with this technology. Six unique methylsite-specific Fabs were identified by initial peptide ELISA and further analyzed by ELISA, surface plasmon resonance (SPR), and Western blot. Further detailed analysis of the antibody–antigen complex for four Fabs was performed by X-ray crystallography and molecular dynamics simulations. Together with the results of functional analyses, we discuss the characteristics of molecular recognition by functional methylsite-specific antibodies. To generate methylsite-specific antibodies, rabbits were immunized with a MAP3K2 peptide sequence surrounding trimethylated Lys260 (Fig. 1, A–B), N-terminally conjugated to keyhole limpet hemocyanin (KLH). After confirmation of a robust and methylation-specific serum response by ELISA, bone marrow and spleen RNA were extracted and a Fab phagemid library was constructed. Fabs were selected by several rounds of biopanning on bovine serum albumin (BSA)-conjugated methylated peptide antigen, with subtraction of nonspecific binders by including an excess of soluble nonmethylated peptide in solution during the binding step (Fig. 1C). Screening of selected Fab clones was carried out by soluble Fab-ELISA (Fig. S1). The absorbance derived from the binding to the BSA-conjugated methylated peptide was compared with that of the unconjugated nonmethylated peptide, and the DNA sequences of methyl-specific clones were analyzed. The Fab clones were divided into six clusters (Fig. 1D) based on the identities of the CDR H3 sequences, and six Fab clones, named E10, F9, C9, E7, E6, and D6, from each cluster that contained more than two unique sequences (clusters 1–5) were chosen for further analysis. The ELISA signal for each clone is summarized in Table S1, and the CDR sequences (based on Chothia definition) of select clones are given in Figure 1E. It is notable that despite the Fabs recognizing the same antigen, each of the Fabs characterized in detail possesses a unique CDR L3 sequence of 10 to 13 amino acids in length and a unique CDR H3 sequence (with the exception of two clones sharing identical H3 sequences) of 5, 6, or 9 amino acids in length. To evaluate the specificity against methylation, we prepared each antibody as a Fab construct, which consists of the antigen-binding domain in antibodies (15Vidarsson G. Dekkers G. Rispens T. IgG subclasses and allotypes: from structure to effector functions.Front. Immunol. 2014; 5: 520Crossref PubMed Scopus (942) Google Scholar), as a purified recombinant protein and determined binding affinity as well as kinetic parameters toward both unconjugated trimethylated and nonmethylated peptides by using surface plasmon resonance (SPR) (Fig. 2A and Fig. S2). The KD values toward methylated and nonmethylated peptides for all the Fabs are plotted in Figure 2B, and the binding affinity and kinetic parameters of the interaction are summarized in Table S2. The result showed that F9, C9, E6, and D6 preferentially bound to the methylated peptide as consistent with Fab ELISA. These clones possessed affinities of 3.0 ×102, 14, 0.90, and 54 nM, respectively, for the methylated peptide. Affinities for the nonmethylated peptides were within the millimolar to 0.3-μM range. It is surprising that E10 showed a similar weak binding to both peptides with 40 μM KD affinity, contrary to ELISA. It is intriguing that E7 did not show any significant binding response in SPR, although ELISA indicates a strong binding activity to the methylated peptide. This is presumably a result of the different peptide presentations in the two assays; ELISA used immobilized, BSA-conjugated peptide antigen, and SPR used soluble free peptide antigen (with immobilized Fab). To examine this hypothesis, we prepared the methylated peptide conjugated with another carrier protein human serum albumin and conducted ELISA analysis. The result showing that E7 bound to the conjugated peptide (Fig. S3) would support the above hypothesis that E7 recognized not solely the peptide sequence but the local structure induced by conjugation with carrier proteins. Furthermore, as MAP3K2 Lys260 can also be mono- and di-methylated (16Van Aller G.S. Graves A.P. Elkins P.A. Bonnette W.G. McDevitt P.J. Zappacosta F. Annan R.S. Dean T.W. Su D.S. Carpenter C.L. Mohammad H.P. Kruger R.G. Structure-based design of a novel SMYD3 inhibitor that bridges the SAM-and MEKK2-binding pockets.Structure. 2016; 24: 774-781Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), we conducted SPR analysis using mono- and di-methylated peptides to evaluate the specificity of each antibody toward the methylation state of the peptide. The result showed that the affinity increased with greater methylation degree, although this dependency was not identical among the antibodies (Table S3). These results suggest that, although the antibodies also recognize the mono- and di-methylated antigen, they have some specificity (especially for C9 and D6) toward the degree of methylation. To assess the sequence specificity for the Fabs possessing high affinity to the methylated peptide, F9, C9, E6, and D6 were tested by SPR using a histone H4 peptide containing K5 trimethylation, which possess three consensus amino acid sequences surrounding the methylation site with MAP3K2 (GK(me3)GG). Although C9 and D6 showed detectable binding to the H4 peptide, the affinity was significantly lower than that toward the target peptide (Fig. 2C). In addition, C9 and D6 showed essentially no affinity (approximately millimolar level) to a peptide derived from histone H3 containing a trimethylated K27, which possess different amino acids sequences surrounding the trimethylated lysine (Fig. S4). These results suggest that these Fabs are not only methylated lysine specific but are also methylsite specific, as they have a strong specificity toward methylated lysine in the context of MAP3K2 surrounding amino acids. We next sought to test whether these Fabs recognize methylated protein antigen in the context of other cellular proteins. We converted the clones to rabbit IgG format for Western blot analysis. HEK293 cells were transfected with a MAP3K2 expression vector together with either a mock or SMYD3 expression vector, as SMYD3 upregulation has been shown to methylate Lys260 of MAP3K2 (13Colón-Bolea P. Crespo P. Lysine methylation in cancer: SMYD3-MAP3K2 teaches us new lessons in the Ras-ERK pathway.Bioessays. 2014; 36: 1162-1169Crossref PubMed Scopus (26) Google Scholar, 14Mazur P.K. Reynoird N. Khatri P. Jansen P.W.T.C. Wilkinson A.W. Liu S. Barbash O. Van Aller G.S. Huddleston M. Dhanak D. Tummino P.J. Kruger R.G. Garcia B.A. Butte A.J. Vermeulen M. et al.SMYD3 links lysine methylation of MAP3K2 to Ras-driven cancer.Nature. 2014; 510: 283-287Crossref PubMed Scopus (232) Google Scholar). Both MAP3K2 and SMYD3 expressions were confirmed by analysis of HA or FLAG tags, respectively (Fig. 3A). Each IgG clone was then tested by Western blot (Fig. 3B). Two off-target bands appeared on staining with F9, neither of which corresponds to the expected size of MAP3K2 and the intensity of which is not dependent on SMYD3 expression. This result indicates that F9 does not appear to recognize protein antigen. On the contrary, the band corresponding to MAP3K2 was clearly shown by staining with C9 and D6 in a SMYD3 expression-dependent manner. Although some bands, which might be derived from other trimethylated proteins that possess similar amino acids surrounding methylated lysine, were also observed in a SMYD3 expression-dependent manner, the band derived from MAP3K2 was dominant, guaranteeing the specificity of the antibodies. Furthermore, to verify that these antibodies indeed recognized the methylated Lys260, detection using the MAP3K2 K260A mutant was tested. The corresponding band was significantly diminished by the mutation for both C9 and D6 (Fig. 3C), demonstrating that these antibodies specifically recognize methylated Lys260 residue. It is intriguing that E6 staining only showed a faint band despite possessing the highest affinity among the Fabs toward the methylated peptide. These results indicate that antigen affinity alone is not predictive of antibody behavior in other applications such as Western blotting, especially when the application involves full-length protein antigen. To gain further insight into how the antibodies recognize the antigen, we determined the crystal structures of each Fab–methylated peptide complex. Omit electron density maps of the peptides indicate with a high degree of confidence that these peptides are bound to the antibodies in the crystal (Fig. S5). Antibodies F9 and E6 engage the peptide predominantly using their heavy chain, with the methylated lysine pointing toward the light chain (Fig. 4A). These similar binding modes could be expected, as the heavy chain CDRs of F9 and E6 differ by only a single amino acid in CDRs H1 and H2 (Fig. 1E). On the other hand, the methylated lysine residue was deeply buried in the interface between the heavy and light chains in C9 and in D6, both of which recognized the protein antigen in Western blotting. Indeed, the calculated accessible surface area of the methylated lysine residue side chain in each complex (F9, 45.3 Å2; C9, 3.8 Å2; E6, 34.0 Å2; and D6, 0.0 Å2) clearly demonstrates the nearly complete penetration of the methylated lysine residue into the interchain cleft for C9 and D6. Of importance, both the N-terminal and C-terminal portions of the peptide were not observed in the complex suggesting that each terminus is disordered. This binding mode may be compatible with antigen recognition in the context of the intact protein, as would occur in Western blotting. Meanwhile, the full C terminus of the peptide was observed in structures of the complex in other Fabs. By studying the local environment surrounding the methylated lysine residues, we found that the four antibody clones examined recognize the methylated lysine residue by using several aromatic residues in a cooperative manner (Fig. 4B). The pockets formed with the aromatic residues, termed “aromatic cage,” are also observed in reader proteins working in the recognition of histone modifications and an antimethylated histone antibody (10Hattori T. Lai D. Dementieva I.S. Montaño S.P. Kurosawa K. Zheng Y. Akin L.R. Świst-Rosowska K.M. Grzybowski A.T. Koide A. Krajewski K. Strahl B.D. Kelleher N.L. Ruthenburg A.J. Koide S. Antigen clasping by two antigen-binding sites of an exceptionally specific antibody for histone methylation.Proc. Natl. Acad. Sci. U. S. A. 2016; 113: 2092-2097Crossref PubMed Scopus (19) Google Scholar), which specifically recognize methylated lysine residues (17Taverna S.D. Li H. Ruthenburg A.J. Allis C.D. Patel D.J. How chromatin-binding modules interpret histone modifications: lessons from professional pocket pickers.Nat. Struct. Mol. Biol. 2007; 14: 1025-1040Crossref PubMed Scopus (1099) Google Scholar). These structures strongly suggest that these Fabs acquired their specificity toward methylation by using the same structural principles. Considering that each Fab uses different combinations of aromatic residues, i.e., F9 uses two Tyr and one Trp, C9 uses two Phe and one Tyr, E6 uses three Tyr, and D6 uses three Trp, the combination of aromatic residue is not likely to be a fundamental for the recognition of methylated lysine residue, instead indicating the general importance of the CH–pi interaction (18Nishio M. Umezawa Y. Hirota M. Takeuchi Y. The CH/π interaction: significance in molecular recognition.Tetrahedron. 1995; 51: 8665-8701Crossref Scopus (578) Google Scholar) between the aromatic residues and methyl groups in the methylated lysine. Looking at the entire peptide in the complexes, each Fab also made hydrogen bonds as well as salt bridges with other parts of the peptide. All the hydrogen bonds and salt bridges, calculated by PDBePISA (19Krissinel E. Henrick K. Inference of macromolecular assemblies from crystalline state.J. Mol. Biol. 2007; 372: 774-797Crossref PubMed Scopus (5906) Google Scholar), are summarized in Supplementary Note 1. F9 and E6 each engaged in 17 hydrogen bonds and 4 salt bridges with the methylated peptide (Fig. S6). On the other hand, C9 and D6 each made seven hydrogen bonds with the peptide (Fig. S6). The fewer hydrogen bonds observed in C9 and D6 compared with F9 and E6, together with the comparable affinity ranges among the Fabs, also suggests the large contribution that deeply located aromatic cages play in peptide binding. Of importance, F9 and E6 engaged in several hydrogen bonds as well as salt bridges with the peptide’s C-terminal carboxyl group, which may explain why these Fabs cannot recognize the full-length protein. To confirm the contribution of these noncovalent bonds with the C-terminal carboxyl group, we conducted SPR analysis using a methylated peptide in which the C terminus was amidated (Fig. S7). The results show that the binding response of the amidated peptide was nearly abolished for F9 and E6, whereas in contrast C9 and D6 recognized the amidated peptide with comparable affinity with that of the nonamidated peptide. This demonstrates that the major reason that F9 and E6 do not recognize protein antigen is their bonding to the C-terminal carboxyl group, which would be incompatible with binding in the context of the full protein sequence. To further characterize the recognition of the methylated lysine residue by the aromatic cages and to explore the possibility of in silico prediction of specificity toward methylation for each antibody, we conducted multiple 400-ns molecular dynamics simulations for each Fab–peptide complex. First, we calculated the RMSD values of the NZ atoms of the methylated lysine after superposing the side-chain atoms of aromatic residues in each structure. The RMSD plots indicate that the methylated lysine residue was stably bound by the aromatic cage in C9, E6, and D6, whereas it dynamically moved in the F9 complex (Fig. 5A). Of importance, the F9 structure showed that the methylated lysine was completely thrown out from the cage at several time points during the simulations, and the antibody retained the peptide only through interaction with the C terminus (Fig. S8). This exclusion of the methylated lysine residue from the aromatic cage was also confirmed by the calculation of the solvent accessible surface area of the methylated lysine residue (Fig. S9). This result appears to be concordant with the SPR results, which showed that C9, E6, and D6 have higher preference toward methylated peptide compared with F9, indicating that our simulations appropriately recaptures antigen recognition observed in the in vitro experiments. We subsequently evaluated the stability of the aromatic cage formation in C9 andD6, both of which recognized protein antigen, to assess if the cages stably form regardless of the antigen. We calculated RMSD values of the all the carbon and nitrogen atoms of the three aromatic residues that form the cages after superposing all the Cα atoms of each complex for both the Fab–methylated peptide complex and apo-Fabs, which were computationally generated based on the crystal structures of the complexes (Fig. 5B). The results showing large fluctuation of RMSD values in the apo structures indicate that the relative position of aromatic residues to each other continuously changed; i.e., the cage collapsed in the absence of peptide antigen, suggesting that the aromatic cages would stably form only in the presence of the antigen even in the C9 and D6 antibodies. In the present study, we created antibodies that recognize the peptide antigen in a methylation-dependent manner by using rabbit immunization followed by antibody library construction and phage display-based selection. This is the first report of detailed structural characterization of antibodies elicited by the use of this novel technique that pairs rabbit immunization with phage display. It is worth noting that, to the best of our knowledge, this is also the first study to characterize the structural and biophysical basis for recognition of a protein antigen by a series of methylation-specific antibodies. Rabbit monoclonal antibodies (mAbs) generally possess higher affinity (picomolar versus nanomolar KD) over their mouse and human counterparts (20Borras L. Gunde T. Tietz J. Bauer U. Hulmann-Cottier V. Grimshaw J.P.A. Urech D.M. Generic approach for the generation of stable humanized single-chain Fv fragments from rabbit monoclonal antibodies.J. Biol. Chem. 2010; 285: 9054-9066Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) owing to extensive somatic hypermutation and even somatic gene conversion, a mechanism absent in humans and mice (21Winstead C.R. Zhai S.K. Sethupathi P. Knight K.L. Antigen-induced somatic diversification of rabbit IgH genes: gene conversion and point mutation.J. Immunol. 1999; 162: 6602-6612PubMed Google Scholar, 22Strohl W. Antibody discovery: sourcing of monoclonal antibody variable domains.Curr. Drug Discov. Technol. 2014; 11: 3-19Crossref PubMed Scopus (28) Google Scholar). Owing to an increased CDR3 length and sequence diversity, recombinant rabbit MAbs have been shown to provide high-quality detection for difficult epitopes including PTM such as phosphorylation and methylation, especially when MAbs from other species had failed (23Chen Y. Vaine M. Wallace A. Han D. Wan S. Seaman M.S. Montefiori D. Wang S. Lu S. A novel rabbit monoclonal antibody platform to dissect the diverse repertoire of antibody epitopes for HIV-1 Env immunogen design.J. Virol. 2013; 87: 10232-10243Crossref PubMed Scopus (24) Google Scholar). However, traditional rabbit mAb platforms rely on hybridoma technology or B cell cloning to generate lead candidates and these systems suffer from limited library sizes (104–106, compared with 1010–1011 in phage display). Also, clone recovery by single cell PCR can be inefficient, causing loss of important lead candidates. Alternatively, historical rabbit phage display systems have failed to compensate for the unique disulfide architecture of rabbit Fabs that can cause expression problems in Escherichia. coli, where the most commonly used Ck1 chain possesses an additional disulfide bond that is not found in mouse or human kappa chains. This led to the development of suboptimal systems that are limited to the underutilized Ck2 chain and thus do not use the full rabbit immune repertoire. To overcome these challenges, we used a patented library construction method paired with an optimized proprietary Fab–phage display vector (containing a hairpin structure to prevent transcription by leaky lac-promoter and strictly controlling the production of Fabs in native state) that allows for display of fully rabbit Fab in the natural form without needing to rely on chimeric alternatives. Rabbit antibody genes" @default.
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• W3111907453 title "Structural basis for antigen recognition by methylated lysine–specific antibodies" @default.
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