Epitope mapping of a monoclonal antibody directed against the α‐subunit of phosphofructokinase‐1 from Saccharomyces cerevisiae by screening phage display libraries

Phosphofructokinase‐1 from Saccharomyces cerevisiae is composed of two types of subunits, α and β. Subunit‐specific monoclonal antibodies were raised to elucidate structural and functional properties of both subunits. One monoclonal antibody, α‐F3, binds to an epitope either at the C‐terminal or at the N‐terminal part of the α‐polypeptide chain. By screening a heptapeptide library with this monoclonal antibody, a set of heptapeptides was selected, which contained the consensus sequences D–A–F and D–S–F. Two heptapeptides with these motifs were synthesized in order assess their capacity to inhibit the binding of antibody α‐F3 to native phosphofructokinase‐1. The peptide G–I–K–D–A–F–L inhibited the binding more strongly (IC₅₀ = 1.5 µM) than the peptide A–P–W–H–D–S–F (IC₅₀ = 33.3 µM). Sequence matching revealed the presence of the D–A–F motif in the polypeptide chain of phosphofructokinase‐1 at amino acid position 172–174. As a control, the nonapeptide A–P–T–S–K–D–A–F–L which corresponds to the sequence of the putative epitope was tested in the inhibition assay. In view of the high inhibitory capacity (IC₅₀ = 0.3 µM) it was concluded that this nonapeptide represents the continuous epitope of phosphofructokinase‐1 that is recognized by antibody α‐F3. Copyright © 1999 John Wiley & Sons, Ltd.     

Characterization of continuous B‐cell epitopes in the N‐terminus of glutamate decarboxylase67 using monoclonal antibodies

Glutamate decarboxylase (GAD) is an autoantigen associated with the autoimmune disorders Type‐1 diabetes (T1D) and stiff‐person syndrome (SPS). The protein, being an essential enzyme involved in the production of the inhibitory neurotransmitter γ‐aminobutyric acid, exists in two isoforms, GAD67 and GAD65. Both isoforms may be targeted by autoantibodies in SPS and T1D patients, although SPS primarily is associated with the presence of GAD67 autoantibodies, whereas T1D mainly is associated with the presence of GAD65 autoantibodies. In this study, we describe antibody reactivity to overlapping GAD67 peptides covering the complete protein sequence by modified peptide enzyme‐linked immunosorbent assay in order to identify potential GAD67 epitopes using two monoclonal antibodies (mAbs). Both GAD67 mAbs showed reactivity to linear epitopes located at the N‐terminal end of GAD67. The epitopes of GAD mAb 1 and 2 were identified as the amino acid sequences NAGADPNTTN and TETDFSNLF, respectively, corresponding to amino acids 14–23 and 91–99. Fine mapping of the epitopes revealed that antibody reactivity was related to amino acid side‐chain functionality, rather than amino acid side‐chain specificity. Additionally, results suggested that non‐contact amino acids in the epitope structure were essential for antibody reactivity. The exact role of these amino acids remains to be determined, but they are thought to be involved in backbone hydrogen bonds or stabilization of the epitope structure. As only limited knowledge is available in relation to antigenic regions of GAD67, this study contributes to characterization of GAD67 epitopes and may be a first step in the development of peptide‐based therapeutics against SPS. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

A model of a gp120 V3 peptide in complex with an HIV-neutralizing antibody based on NMR and mutant cycle-derived constraints.

The 0.5beta monoclonal antibody is a very potent strain-specific HIV-neutralizing antibody raised against gp120, the envelope glycoprotein of HIV-1. This antibody recognizes the V3 loop of gp120, which is a major neutralizing determinant of the virus. The antibody-peptide interactions, involving aromatic and negatively charged residues of the antibody 0.5beta, were studied by NMR and double-mutant cycles. A deuterated V3 peptide and a Fab containing deuterated aromatic amino acids were used to assign these interactions to specific V3 residues and to the amino acid type and specific chain of the antibody by NOE difference spectroscopy. Electrostatic interactions between negatively charged residues of the antibody Fv and peptide residues were studied by mutagenesis of both antibody and peptide residues and double-mutant cycles. Several interactions could be assigned unambiguously: F96(L) of the antibody interacts with Pro13 of the peptide, H52(H) interacts with Ile7, Ile9 and Gln10 and D56(H) interacts with Arg11. The interactions of the light-chain tyrosines with Pro13 and Gly14 could be assigned to either Y30a(L) and Y32(L), respectively, or Y32(L) and Y49(L), respectively. Three heavy-chain tyrosines interact with Ile7, Ile20 and Phe17. Several combinations of assignments involving Y32(H), Y53(H), Y96(H) and Y100a(H) may satisfy the NMR and mutagenesis constraints, and therefore at this stage the interactions of the heavy-chain tyrosines were not taken into account. The unambiguous assignments [F96(L), H52(H) and D56(H)] and the two possible assignments of the light-chain tyrosines were used to dock the peptide into the antibody-combining site. The peptide converges to a unique position within the binding site, with the RGPG loop pointing into the center of the groove formed by the antibody complementary determining regions while retaining the beta-hairpin conformation and the type-VI RGPG turn [Tugarinov, V., Zvi, A., Levy, R. & Anglister, J. (1999) Nat. Struct. Biol. 6, 331-335].     

Immunochemical Features of Complementarity Determining Region (CDR) Peptide in Anti Hemin Monoclonal Antibody

Messenger RNA purified from the anti hemin monoclonal antibody (1D3) secreting hybridoma was amplified by RT‐PCR and the nueleotide and amino acid sequences of the antibody were determined. The role of complementarity determining regions (CDRs) in porphyrin recognition and its immunochemical feature of the antibody were investigated by using ELISA, fluorescence measurement and computational calculation of the conformation. All CDR peptides of the heavy chain of the antibody were synthesized and their affinity constants to porphyrins were determined. The value of CDR2 of heavy chain (CDRH2) of 1D3 was 1.5×10⁵/M for protoporphyrin and 7×10⁷/M for TCPP, respectively, while that of the whole antibody showed to be 1.2×10⁷/M for TCPP. Though CDRH2 is a 17 meric peptide, it showed higher affinity than the whole antibody (1D3). Porphyrins can be considered to firmly bind with CDRH2, while CDRH3 is not involved in the antigen binding. CDR‐1 may participate in the recognition with a small contribution. By the computational analysis of steric conformation, it was suggested that CDRH1 and CDRH2 co‐operatively function in the recognition of porphyrin. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Detecting low level sequence variantsin recombinant monoclonal antibodies

A systematic analytical approach combining tryptic and chymotryptic peptide mapping with a Mascot Error Tolerant Search (ETS) has been developed to detect and identify low level protein sequence variants, i.e., amino acid substitutions, in recombinant monoclonal antibodies. The reversed-phase HPLC separation with ultraviolet (UV) detection and mass spectral acquisition parameters of the peptide mapping methods were optimized by using a series of model samples that contained low levels (0.5–5.0%) of recombinant humanized anti-HER2 antibody (rhumAb HER2) along with another unrelated recombinant humanized monoclonal antibody (rhumAb A). This systematic approach’s application in protein sequence variant analysis depends upon time and sensitivity constraints. An example of using this approach as a rapid screening assay is described in the first case study. For stable CHO clone selection for an early stage antibody project, comparison of peptide map UV profiles from the top four clone-derived rhumAb B samples quickly detected two sequence variants (M83R at 5% and P274Tat 42% protein levels) from two clones among the four. The second case study described in this work demonstrates how this approach can be applied to late stage antibody projects. A sequence variant, L413Q, present at 0.3% relative to the expected sequence of rhumAb C was identified by a Mascot-ETS for one out of four top producers. The incorporation of this systematic sequence variant analysis into clone selection and the peptide mapping procedure described herein have practical applications for the biotechnology industry, including possible detection of polymorphisms in endogenous proteins.Key words: recombinant monoclonal antibody, cell line development, sequence variants, HPLC-UV/MS/MS, tryptic peptide mapping, Mascot error tolerant search     

Comparison of the amino acid sequences of the variable domains of two homogeneous rabbit antibodies to type III pneumococcal polysaccharide.

The amino acid sequences of the V (variable) regions of the H (heavy) and L (light) chains derived from rabbit antibody K-25, specific for type III pneumococci, were determined; this is the second homogeneous rabbit antibody besides antibody BS-5 whose complete sequence of the V domain has been established (Jaton, 1974d). The V regions of L chains BS-5 and K-25 (both of allotype b4) differ from each other by 19 amino acid residues; 11 of these 19 substitutions are located within the three hypervariable sections of the V region. On the basis of seven amino acid differences within the N-terminal 28 positions, it is suggested that L chain K-25 belongs to a different subgroup of rabbit K chains and L chain BS-5. H chain K-25 (allotype a2) differs from another H chain of the same allotype by one amino acid substitution within the N-terminal 70 positions in addition to interchanges occurring in the first two hypervariable sections. H chain K-25 was compared with H chain BS-5 (allotype a1) and with the known V-region rabbit sequences. Allotype-related differences between a1, a2 and a3 chains appear to occur within the N-terminal 16 positions and possibly in scattered positions throughout the V-region. In the hypervariable positions, variability between the two antibodies is remarkably more pronounced within the third hypervariable section of both H and L chains than within the first two.     

Rapid comparison of a candidate biosimilar to an innovator monoclonal antibody with advanced liquid chromatography and mass spectrometry technologies

This study shows that state-of-the-art liquid chromatography (LC) and mass spectrometry (MS) can be used for rapid verification of identity and characterization of sequence variants and posttranslational modifications (PTMs) for antibody products. A candidate biosimilar IgG1 monoclonal antibody (mAb) was compared in detail to a commercially available innovator product. Intact protein mass, primary sequence, PTMs, and the micro-differences between the two mAbs were identified and quantified simultaneously. Although very similar in terms of sequences and modifications, a mass difference observed by LC-MS intact mass measurements indicated that they were not identical. Peptide mapping, performed with data independent acquisition LC-MS using an alternating low and elevated collision energy scan mode (LC-MS^E), located the mass difference between the biosimilar and the innovator to a two amino acid residue variance in the heavy chain sequences. The peptide mapping technique was also used to comprehensively catalogue and compare the differences in PTMs of the biosimilar and innovator mAbs. Comprehensive glycosylation profiling confirmed that the proportion of individual glycans was different between the biosimilar and the innovator, although the number and identity of glycans were the same. These results demonstrate that the combination of accurate intact mass measurement, released glycan profiling, and LC-MS^E peptide mapping provides a set of routine tools that can be used to comprehensively compare a candidate biosimilar and an innovator mAb.     

The testosterone binding mechanism of an antibody derived from a naïve human scFv library

A testosterone binding scFv antibody was isolated from a naïve human library with a modest size of 10⁸ clones. The crystal structure of the Fab fragment form of the 5F2 antibody clone complexed with testosterone determined at 1.5 Å resolution shows that the hapten is bound deeply in the antibody binding pocket. In addition to the interactions with framework residues only CDR‐L3 and CDR‐H3 loops interact with testosterone and the heavy chain forms the majority of the contacts with the hapten. The testosterone binding site of the 5F2 antibody with a high abundance of aromatic amino acid residues shows similarity with an in vitro affinity matured antibody having around 300 times higher affinity. The moderate affinity of the 5F2 antibody originates from the different orientation of the hapten and few light chain contacts. This is the first three‐dimensional structure of a human steroid hormone binding antibody that has been isolated from a naïve human repertoire. Copyright © 2010 John Wiley & Sons, Ltd.     

Detecting low level sequence variants in recombinant monoclonal antibodies

A systematic analytical approach combining tryptic and chymotryptic peptide mapping with a Mascot Error Tolerant Search (ETS) has been developed to detect and identify low level protein sequence variants, i.e., amino acid substitutions, in recombinant monoclonal antibodies. The reversed-phase HPLC separation with ultraviolet (UV) detection and mass spectral acquisition parameters of the peptide mapping methods were optimized by using a series of model samples that contained low levels (0.5-5.0%) of recombinant humanized anti-HER2 antibody (rhumAb HER2) along with another unrelated recombinant humanized monoclonal antibody (rhumAb A). This systematic approach’s application in protein sequence variant analysis depends upon time and sensitivity constraints. An example of using this approach as a rapid screening assay is described in the first case study. For stable CHO clone selection for an early stage antibody project, comparison of peptide map UV profiles from the top four clone-derived rhumAb B samples quickly detected two sequence variants (M83R at 5% and P274T at 42% protein levels) from two clones among the four. The second case study described in this work demonstrates how this approach can be applied to late stage antibody projects. A sequence variant, L413Q, present at 0.3% relative to the expected sequence of rhumAb C was identified by a Mascot-ETS for one out of four top producers. The incorporation of this systematic sequence variant analysis into clone selection and the peptide mapping procedure described herein have practical applications for the biotechnology industry, including possible detection of polymorphisms in endogenous proteins.     

Enzymatic ligation for synthesis of single‐chain analogue of monellin by transglutaminase*

Monellin, a sweet protein, consists of two noncovalently associated polypeptide chains: an A chain of 44 amino acid residues and a B chain of 50 residues. Microbial transglutaminase (MTGase) was used for ligation of the monellin subunits without any protecting groups, and without activation of the C^α‐carboxyl group at the C‐terminus. Since a peptide fragment LLQG is a good substrate for MTGase to form an amide bond between the γ‐amide group of the Gln residue and the ε‐amino group of Lys, a monellin B chain analogue in which LLQG was elongated at the C‐terminus (B‐LLQG) was synthesized by solid‐phase synthesis. The monellin A chain analogue in which KGK was elongated at the N‐terminus (KGK‐A) was synthesized by the same method as that of the B chain analogue. The KGK‐A chain and the B‐LLQG chain were coupled by MTGase to give single‐chain analogue of monellin. The single‐chain analogue of monellin was characterized by analytical reverse phase high performance liquid chromatography, electrospray ionization, and amino acid analyses. All analyses gave satisfactory results. The single‐chain analogue of monellin was more heat stable than natural monellin. © 1999 John Wiley & Sons, Inc. Biopoly 50: 193–200, 1999     

从抗TNF抗体的CDR肽库中获得拮抗TNF的短肽

为设计来自抗体的短肽 ,以抗肿瘤坏死因子 (TNF)嵌合抗体 (cA2 )CDRs为模板 ,在其两侧各加 3个随机氨基酸残基 ( X3 CDR X3 ) ,构建了 6个以CDR为基础的肽库 .经过 3轮亲和选择 ,挑取单克隆 ,进一步经ELISA检测TNF阳性噬菌体克隆 ,分离得到 7个ELISA阳性较好的噬菌体肽克隆 ,分别命名为CDR2L1、CDR2L2、CDR2L3、CDR1L1、CDR2H1、CDR3H1、CDR3H 2 .应用MTT方法 ,检测 7个克隆对TNF生物学活性的拮抗作用 .结果显示 :来自CDR2L ,CDR3H肽库中的CDR2L2、CDR2L3,CDR3H2噬菌体肽具有明显的拮抗TNF诱导L92 9细胞的细胞毒作用 ,其中以CDR2L2噬菌体肽的拮抗活性最强 .而来源于CDR1L ,CDR2H肽库的CDR1L1和CDR2H1噬菌体肽和来自CDR2L ,CDR3H肽库中的CDR2L1和CDR3H1噬菌体肽没有明显的拮抗TNF作用 .研究结果初步表明 :从cA2抗体CDR肽库中筛选得到的噬菌体CDR模拟肽具有亲本抗体相似的结合活性和生物学效应 ,从而为开发已知抗体 (特别是治疗用抗体 )CDR为基础的肽药物创建一个技术平台奠定基础     

Multispecificity of a recombinant anti‐ras monoclonal antibody

Recombinant monoclonal antibodies (Ab's) have widespread application as research tools, diagnostic reagents and as biotherapeutics. Whilst studying the cellular molecular switch protein m‐ras, a recombinant monoclonal antibody to m‐ras was generated for use as a research tool. Antibody genes from a single rabbit B cell secreting IgG to an m‐ras specific peptide sequence were expressed in mammalian cells, and monoclonal rabbit IgG binding was characterized by ELISA and peptide array blotting. Although the monoclonal Ab was selected for specificity to m‐ras peptide, it also bound to both recombinant full‐length m‐ras and h‐ras proteins. The cross‐reactive binding of the monoclonal Ab to h‐ras was defined by peptide array blot revealing that the Ab showed preference for peptide sequences containing multiple positively charged amino acid residues. These data reinforce the concept of antibody multispecificity through multiple interactions of the Ab paratope with diverse polypeptides. They also emphasize the importance of immunogen and Ab selection processes when generating recombinant monoclonal Ab's.     

Asymmetrically simultaneous synthesis of L‐homophenylalanine and N6‐protected‐2‐oxo‐6‐amino‐hexanoic acid by engineered Escherichia coli aspartate aminotransferase

L ‐Homophenylalanine (L ‐HPA) and N⁶‐protected‐2‐oxo‐6‐amino‐hexanoic acid (N⁶‐protected‐OAHA) can be used as building blocks for the manufacture of angiotensin‐converting enzyme inhibitors. To synthesize L ‐HPA and N⁶‐protected‐OAHA simultaneously from 2‐oxo‐4‐phenylbutanoic acid (OPBA) and N⁶‐protected‐L ‐lysine, several variants of Escherichia coli aspartate aminotransferase (AAT) were developed by site‐directed mutagenesis and their catalytic activities were investigated. Three kinds of N⁶‐protected‐L ‐lysine were tested as potential amino donors for the bioconversion process. AAT variants of R292E/L18H and R292E/L18T exhibited specific activities of 0.70±0.01 U/mg protein and 0.67±0.02 U/mg protein to 2‐amino‐6‐tert‐butoxycarbonylamino‐hexanoic acid (BOC‐lysine) and 2‐amino‐6‐(2,2,2‐trifluoro‐acetylamino)‐hexanoic acid, respectively. E. coli cells expressing R292E/L18H variant were able to convert OPBA and BOC‐lysine to L ‐HPA and 2‐oxo‐6‐tert‐butoxycarbonylamino‐hexanoic acid (BOC‐OAHA) with 96.2% yield in 8 h. This is the first report demonstrating a process for the simultaneous production of two useful building blocks, L ‐HPA and BOC‐OAHA. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Rapid comparison of a candidate biosimilar to an innovator monoclonal antibody with advanced liquid chromatography and mass spectrometry technologies

This study shows that state-of-the-art liquid chromatography (LC) and mass spectrometry (MS) can be used for rapid verification of identity and characterization of sequence variants and posttranslational modifications (PTMs) for antibody products. A candidate biosimilar IgG1 monoclonal antibody (mAb) was compared in detail to a commercially available innovator product. Intact protein mass, primary sequence, PTMs and the micro-differences between the two mAbs were identified and quantified simultaneously. Although very similar in terms of sequences and modifications, a mass difference observed by LC-MS intact mass measurements indicated that they were not identical. Peptide mapping, performed with data independent acquisition LC-MS using an alternating low and elevated collision energy scan mode (LC-MS^E), located the mass difference between the biosimilar and the innovator to a two amino acid residue variance in the heavy chain sequences. The peptide mapping technique was also used to comprehensively catalogue and compare the differences in PTMs of the biosimilar and innovator mAbs. Comprehensive glycosylation profiling confirmed that the proportion of individual glycans was different between the biosimilar and the innovator, although the number and identity of glycans were the same. These results demonstrate that the combination of accurate intact mass measurement, released glycan profiling and LC-MS^E peptide mapping provides a set of routine tools that can be used to comprehensively compare a candidate biosimilar and an innovator mAb.Key words: biosimilar mAb, innovator mAb, molecular similarity, sequence variants, posttranslational modifications, N-linked glycosylation, chemical degradations, micro-heterogeneities, characterization, intact protein mass measurement, peptide mapping, glycan profiling, LC-MS, LC-fluorescence, MALDI MS     

Stable production of cyanophycinase in Nicotiana benthamiana and its functionality to hydrolyse cyanophycin in the murine intestine

Food supplementation with the conditionally essential amino acid arginine (Arg) has been shown to have nutritional benefits. Degradation of cyanophycin (CGP), a peptide polymer used for nitrogen storage by cyanobacteria, requires cyanophycinase (CGPase) and results in the release of β‐aspartic acid (Asp)‐Arg dipeptides. The simultaneous production of CGP and CGPase in plants could be a convenient source of Arg dipeptides. Different variants of the cphB coding region from Thermosynechococcus elongatus BP‐1 were transiently expressed in Nicotiana benthamiana plants. Translation and enzyme stability were optimized to produce high amounts of active CGPase. Protein stability was increased by the translational fusion of CGPase to the green fluorescent protein (GFP) or to the transit peptide of the small subunit of RuBisCO for peptide production in the chloroplasts. Studies in mice showed that plant‐expressed CGP fed in combination with plant‐made CGPase was hydrolysed in the intestine, and high levels of ß‐Asp‐Arg dipeptides were found in plasma, demonstrating dipeptide absorption. However, the lack of an increase in Asp and Arg or its metabolite ornithine in plasma suggests that Arg from CGP was not bioavailable in this mouse group. Intestinal degradation of CGP by CGPase led to low intestinal CGP content 4 h after consumption, but after ingestion of CGP alone, high CGP concentrations remained in the large intestine; this indicated that intact CGP was transported from the small to the large intestine and that CGP was resistant to colonic microbes.     

Identification of peptide mimotopes for the fluorescein hapten binding of monoclonal antibody B13‐DE1

Using 6mer and 12mer phage peptide libraries three unique phage clones were identified which specifically bind to a monoclonal anti‐FITC antibody, B13‐DE1. The two 6mer and one 12mer peptide insert sequences are clearly related to each other and contain a high proportion of hydrophobic amino acids. The peptides are bound by the antibody combining site of B13‐DE1 probably in a similar manner to FITC and represent therefore true peptidic mimics of the fluorescein hapten. No reactivity of the peptides could be demonstrated with another monoclonal anti‐fluorescein antibody or with polyclonal anti‐fluorescein antibodies. Immunization of mice with the peptides resulted in the production of antibodies cross‐reacting with all peptides but not with fluorescein. The results show that phage peptide libraries can be used to isolate mimotope peptides which can mimic low molecular weight structures seen by a specific antibody and probably other recognition molecules. Copyright © 1999 John Wiley & Sons, Ltd.     

Connective tissue microfibrils. Isolation and characterization of three large pepsin-resistant domains of fibrillin

Human amnion was solubilized using pepsin and the digest supernatant screened for fragments of fibrillin with a previously characterized monoclonal antibody (Sakai, L. Y., Keene, D. R., and Engvall, E. (1986) J. Cell Biol. 103, 2499-2509). One fragment (PF1), with an apparent molecular weight of 94,000, was isolated and characterized. Two other fragments, PF2 and PF3, were isolated and shown to be fragments of fibrillin by preparing a monospecific antisera to PF2 and a monoclonal antibody to PF3. Immunoelectron microscopy and immunoblotting showed that both antibodies were specific for fibrillin. Electron microscope pictures of rotary-shadowed PF1 and PF2 showed them to be short rod-shaped molecules while PF3 has a crab-like appearance and seems to be an aggregate of several fibrillin chain fragments. Amino-terminal amino acid sequencing of PF1 and PF2 gave single unique sequences. Each of the three antibodies used was specific for one fragment and peptide mapping of PF1 and PF2 showed that there was no significant amino acid sequence overlap. Aggregates of PF3 are described which provided insight into the assembly and macromolecular structure of fibrillin in microfibrils.     

Characterization by liquid chromatography combined with mass spectrometry of monoclonal anti-IGF-1 receptor antibodies produced in CHO and NS0 cells

7H2HM is a new humanized recombinant monoclonal antibody (MAb) directed against insulin-like growth factor-1 receptor and produced in CHO cells. Homogeneity of intact antibody, reduced light and heavy chains, Fab and Fc fragments were investigated by analytical methods based on mass (SDS-PAGE, SEC), charge (IEF, C-IEX) and hydrophobicity differences (RP-HPLC, HIC) and compared side-by-side with A2CHM, produced in NS0 cells. Primary structures and disulfide bridge pairing were analyzed by microsequencing (Edman degradation), mass spectrometry (MALDI-TOF, ES-TOF) and peptide mapping after enzymatic digestion (Trypsin, endoprotease Lys-C, papain). The light chains demonstrated the expected sequences. The heavy chains yielded post-translational modifications previously reported for other recombinant humanized or human IgG1 such as N-terminal pyroglutamic acid, C-terminal lysine clipping and N-glycosylation for asparagine 297. More surprisingly, two-thirds of the 7H2HM heavy chains were shown to contain an additional 24-amino-acid sequence, corresponding to the translation of an intron located between the variable and the constant domains. Taken together these data suggest that 7H2HM is a mixture of three families of antibodies corresponding (i) to the expected structure (17%; 14,9297 Da; 1330 amino acids), (ii) a variant with a translated intron in one heavy chains (33%; 15,2878 Da; 1354 amino acids) and (iii) a variant with translated introns in two heavy chains (50%; 15,4459 Da; 1378 amino acids), respectively. RP-HPLC is not a commonly used chromatographic method to assess purity of monoclonal antibodies but unlike to SEC and SDS-PAGE, was able to show and to quantify the family of structures present in 7H2HM, which were also identified by peptide mapping, mass spectrometry and microsequencing.     

Epitope mapping of monoclonal antibodies for the Deinococcus radiodurans bacteriophytochome

Bacteriophytochromes (BphP) are phytochrome‐like light sensing proteins in bacteria, which use biliverdin as a chromophore. In order to study the biochemical properties of the DrBphP protein, five (2B8, 2C11, 3B2, 3D2, and 3H7) anti‐DrBphP monoclonal antibodies were produced through the immunization of mice with purified full‐length DrBphP and DrBphN (1–321 amino acid) proteins, and epitope mapping was then carried out. Among the five antibodies, 2B8 and 2C11 preferentially recognized the N‐terminal region of BphP whereas 3B2, 3D2, and 3H7 showed preference for the C‐terminal region. We performed further epitope mapping using recombinant truncated BphP proteins to narrow down their target sequences. The results demonstrated that each of the five monoclonal antibodies recognized different regions on the DrBphP protein. Additionally, epitopes of 2B8 and 3H7 antibodies were discovered to be shorter than 10 amino acids (2B8: RDPLPFFPP, 3H7: PGEIEEA). These two antibodies with such specific recognition epitopes could be especially valuable for developing new peptide tags for protein detection and purification.     

Infrared study of synthetic peptide analogues of the calcium‐binding site III of troponin C: The role of helix F of an EF‐hand motif

The EF‐hand motif (helix–loop–helix) is a Ca²⁺‐binding domain that is common among many intracellular Ca²⁺‐binding proteins. We applied Fourier‐transform infrared spectroscopy to study the synthetic peptide analogues of site III of rabbit skeletal muscle troponin C (helix E–loop–helix F). The 17‐residue peptides corresponding to loop–helix F (DRDADGYIDAEELAEIF), where one residue is substituted by the D ‐type amino acid, were investigated to disturb the α‐helical conformation of helix F systematically. These D ‐type‐substituted peptides showed no band at about 1555 cm^?1 even in the Ca²⁺‐loaded state although the native peptide (L ‐type only) showed a band at about 1555 cm^?1 in the Ca²⁺‐loaded state, which is assigned to the side‐chain COO^? group of Glu at the 12th position, serving as the ligand for Ca²⁺ in the bidentate coordination mode. Therefore, helix F is vital to the interaction between the Ca²⁺ and the side‐chain COO^? group of Glu at the 12th position. Implications of the COO^? antisymmetric stretch and the amide‐I′ of the synthetic peptide analogues of the Ca²⁺‐binding sites are discussed. © 2012 Wiley Periodicals, Inc. Biopolymers 99: 342–347, 2013.