Báo cáo khoa học: Isolation and characterization of an IgNAR variable domain specific for the human mitochondrial translocase receptor Tom70 potx

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Báo cáo khoa học: Isolation and characterization of an IgNAR variable domain specific for the human mitochondrial translocase receptor Tom70 potx

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Isolation and characterization of an IgNAR variable domain specific for the human mitochondrial translocase receptor Tom70 Stewart D. Nuttall 1,2 , Usha V. Krishnan 1,2 , Larissa Doughty 1 , Kylie Pearson 2,3 , Michael T. Ryan 2,3 , Nicholas J. Hoogenraad 2,3 , Meghan Hattarki 1 , Jennifer A. Carmichael 1,2 , Robert A. Irving 1,2 and Peter J. Hudson 1,2 1 CSIRO Health Sciences and Nutrition, and 2 CRC for Diagnostics, Parkville, Victoria, Australia; 3 Department of Biochemistry, La Trobe University, Bundoora, Victoria, Australia The new antigen receptor (IgNAR) from sharks is a disul- phide bonded dimer of two protein chains, each containing one variable and five constant domains, and functions as an antibody. In order to assess the antigen-binding capabilities of isolated IgNAR variable domains (V NAR ), we have con- structed an in vitro library incorporating synthetic CDR3 regions of 15–18 residues in length. Screening of this library against the 60 kDa cytosolic domain of the 70 kDa outer membrane translocase receptor from human mitochondria (Tom70) resulted in one dominant antigen-specific clone (V NAR 12F-11) after four rounds of in vitro selection. V NAR 12F-11 was expressed into the Escherichia coli periplasm and purified by anti-FLAG affinity chromatography at yields of 3mgÆL )1 . Purified protein eluted from gel filtration columns as a single monomeric protein and CD spectrum analysis indicated correct folding into the expected b-sheet confor- mation. Specific binding to Tom70 was demonstrated by ELISA and BIAcore (K d ¼ 2.2 ± 0.31 · 10 )9 M )1 ) indi- cating that these V NAR domains can be efficiently displayed as bacteriophage libraries, and selected against target anti- gens with an affinity and stability equivalent to that obtained for other single domain antibodies. As an initial step in producing ÔintrabodyÕ variants of 12F-11, the impact of modifying or removing the conserved immunoglobulin intradomain disulphide bond was assessed. High affinity binding was only retained in the wild-type protein, which combined with our inability to affinity mature 12F-11, sug- geststhatthisparticularV NAR is critically dependent upon precise CDR loop conformations for its binding affinity. Keywords: new antigen receptor; variable domain; peptide display; Tom70; mitochondrial import. Conventional antibodies recognize antigens through the combination of six complementarity determining region (CDR) loops displayed three each upon variable heavy (V H ) and variable light (V L ) chain immunoglobulin domains [1]. These CDR loops vary in size and composition allowing formation of a large number of conformational antigen- binding surfaces including planar and ridged topologies [2]. The orientation of the loops is maintained by a combination of their internal architecture, the underlying immunoglo- bulin scaffold, and the hydrophobic interaction between the antibody V H and V L domains [3]. In contrast, families of antibody-like molecules characterized recently in camelids and sharks rely on a single immunoglobulin V H -like domain framework, which presents two or three CDR loops to form the antigen-binding interface [4–7]. For camelids, these V H H single domain antibodies can bind an extensive range of antigens, including large proteins, enzymes (either within or outside the active site clefts), haptens and dyes. Biochemical and structural data now shows that V H H binding affinity resides in a variety of possible CDR conformations that can include all three CDRs, or an elongated CDR3 loop alone, or a combination of CDR and framework side-chain and main-chain residues [8]. For sharks, the new antigen receptor (IgNAR) from Ginglymostoma cirratum (nurse sharks) and Orectolobus maculatus (wobbegong sharks) also utilizes a single V H -like domain, which we herein term V NAR [9,10]. Structurally, the entire intact IgNAR antibody molecule is a disulphide- bonded dimer of two protein chains, each containing the single variable and five constant domains. There is no associated light chain and immunoelectron microscopy confirms that the V NAR domains do not associate together across a V H /V L -like interface and thereby provide bivalent affinity to two separate antigen molecules [11]. There is a striking evolutionary convergence at the molecular structure level between the shark V NAR and camelid V H Hantigen- binding domains. Similarities include, but are not limited to (a) the presence of charged rather than hydrophobic residues in the conventional V L interface of the immuno- globulin framework, which imparts a hydrophilic character to the solvent-exposed areas; (b) larger CDR3 loops compared to those found in human and murine antibodies (murine,  XX ¼ 10; human,  XX ¼ 13; llama V H H,  XX ¼ 15; camel V H H,  XX ¼ 17.5; V NAR  XX ¼ 16) [10,12,13]; and Correspondence to Stewart Nuttall, CSIRO Health Sciences and Nutrition, 343 Royal Parade, Parkville, Victoria 3052, Australia. Fax: + 61 3 9662 7314, Tel.: + 61 3 9662 7100, E-mail: Stewart.Nuttall@csiro.au Abbreviations: IgNAR, new antigen receptor antibody from sharks; VNAR, single variable domain of the IgNAR antibody; Tom70, human 70 kDa component of the translocase of the outer mito- chondrial membrane; CDR, complementarity determining region. (Received 14 May 2003, revised 20 June 2003, accepted 1 July 2003) Eur. J. Biochem. 270, 3543–3554 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03737.x (c) the frequent presence of disulphide bridges, indicated by paired cysteine residues, either within the CDR3 loop (Type1 V NAR ) or between the CDR1 and CDR3 loops (Type2 V NAR ) [5,11]. Camelid V H Hs typically possess disulphide linkages either between the CDR1 and )3, or CDR2 and )3 loops [13,14]. Despite these similarities, the camel and shark variable domains are clearly different, both by sequence alignment (only  20% identity) and the unusual focus of shark V NAR variability into only the CDR1 and )3 regions (Type2 V NAR ), or the CDR2 and )3 regions (Type1 V NAR )[7]. While the shark IgNARs have yet to be formally demonstrated as in vivo molecules responsible for immuno- surveillance, there is strong evidence for their functional role in antigen binding. First, an analysis of mutational patterns of membrane-bound and secreted forms of nurse sharkIgNARsindicatedthattheyaremutatedinthelatter form, suggesting affinity maturation by somatic hyper- mutation [15,16]. Second, in a previous study, we showed that the individual wobbegong V NAR s could be expressed as soluble single molecules in the E. coli periplasm. An in vitro Type2 V NAR library was then designed with randomized CDR3 loops, and displayed successfully on the surface of fd bacteriophages and panned for specific binding molecules [10]. Third, and most recently we isolated two naturally occurring V NAR domains targeting the kgp protease from Porphyromonas gingivalis with affinities within the nanomolar range [17]. Finally, a third type of V NAR (Type3) has very recently been identified in neonatal shark primary lymphoid tissue that probably functions as a protective low-specificity antibody early in development, prior to maturation of the Type 1/2 IgNAR antigen-driven response [7]. The Type3 V NAR topology is characterized by a constant sized CDR3 loop of limited diversity, probably stabilized by a conserved tryptophan residue within the CDR1 loop [7]. Here we describe the design and construction of an expanded in vitro V NAR library with more extensive synthetic CDR3 loop variations, and hypothesize that this library should contain binding reagents to a wide range of protein targets. We have chosen as one target antigen the cytosolic domain of the 70 kDa outer membrane trans- locase (Tom70) from human mitochondria that is impli- cated in mitochondrial import processes [18,19]. Studies in Saccharomyces cerevisiae and the fungus, Neurospora crassa have characterized Tom70 as a receptor peripheral to the mitochondrial general insertion pore that preferentially interacts with a subset of preproteins that typically contain internal targeting signals and/or require the action of cytosolic chaperones for their delivery to the mitochondrial surface [20,21]. The human homologue of Tom70 has only recently been identified [22], and attempts to generate high specificity polyclonal or monoclonal antibodies have so far been unsuccessful, perhaps due to extensive sequence homology across species. Here, we report the isolation and characterization of a V NAR that binds with high affinity to human Tom70 as an important demonstration that V NAR libraries can provide novel binding reagents against refractory and immunosilent targets. Further, to demon- strate the effect of removing the internal stabilizing disul- phide bond in the manner of antibody V-domain ÔintrabodiesÕ [23], residues Cys22 and Cys82 were modified by alkylation, or replaced with alanine and valine, respect- ively, and the resultant V NAR s evaluated for retention of binding affinity. Material and methods Equipment and reagents Restriction enzymes and Vent DNA polymerase were purchased from New England Biolabs; T4 DNA ligase was from Biotech (Australia). DNA fragment recovery and purification was by QIAquick Gel Extraction Kit, Qiagen and small-scale preparations of DNA from E. coli were performed using the QIAprep Spin Miniprep Kit, Qiagen. Monoclonal anti-(FLAG) Ig affinity resin was produced as previously described [17]. BenchMark TM Prestained Protein Ladder Cat. # 10748–010 was from Gibco BRL Life Technologies. Standard molecular biological techniques were performed as described [24]. Goat anti-(mouse) IgG (Fc)-HRP was from Pierce. E. coli strains The cell line used for library propagation and selection and protein expression was E. coli TG1 (K12 supE D(lac- proAB) thi hsdD5F¢{traD36 proAB + lacI q lacZDM15]. E. coli transformants were maintained and grown in 2 · YT broth supplemented with 100 lgÆmL )1 (w/v) ampi- cillin and/or 2% (w/v) glucose. Solid media contained 2% (w/v) Bacto-agar. Transformation of E. coli was by stand- ard procedures [24] performed using electrocompetent cells. Isolation of total RNA from Wobbegong sharks Spotted Wobbegong sharks (Orectolobus maculatus)were housed and maintained at the Underwater World Aquar- ium, Mooloolaba, Queensland, Australia. For isolation of peripheral blood lymphocytes, a blood sample (3 mL) was taken from the caudal vein of a young male (6.82 kg). Experiments were performed in accordance with CSIRO (Health Sciences and Nutrition) animal ethics requirements. Total RNA was extracted using the AquaPure RNA Isolation Kit (BIO-RAD, Australia), stored at )80 °C, and used in reverse transcription-polymerase chain reac- tions using the Titan one tube RT-PCR system (Roche, Germany) as described [10]. Library construction and panning DNA library cassettes encoding the Wobbegong V NAR with randomization of the CDR3 loop were constructed as described [10], using CDR3 randomization oligonucleotide primers to generate synthetic CDR3s of 15 residues (#6981); 16 residues (#7211); 17 residues (#6980) and 18 residues (#7210) in length (Table 1). In addition, natural Wobbe- gong V NAR sequences were amplified direct from cDNA using only the variable domain terminal primers (Table 1) [10,17]. Cassette fragments were cut with the restriction endonucleases NotIandSfiI and ligated into similarly cut phagemid display vector, pFAB5c.His [25]. Library liga- tions were purified, pooled and transformed into E. coli TG1, yielding a total library size of approximately 4.0 · 10 8 3544 S. D. Nuttall et al. (Eur. J. Biochem. 270) Ó FEBS 2003 independent clones, which included a subset ( 7 · 10 6 )of clones derived from natural CDR3 sequences. Phagemid particles carrying the V NAR –gene3protein fusion were propagated and isolated by standard procedures [26]. For biopanning of the phagemid library, recombinant human Tom70 (5 lgÆmL )1 in NaCl/P i ) was coated onto Maxisorb Immunotubes and incubated at 4 °C overnight. Immuno- tubes were rinsed (NaCl/P i ), blocked with NaCl/P i /Blotto (2%, w/v; Diploma skim milk powder, Bonlac Foods Ltd., Melbourne, Australia) for 1 h, and incubated with freshly prepared phagemid particles in NaCl/P i /Blotto (2%, w/v) for 30 min at room temperature with gentle agitation followed by 90 min without agiation. After incubation, immunotubes were washed [NaCl/P i /Tween20 (0.1%, v/v); 7, 8, 10 and 10 washes for panning rounds 1–4], followed by an identical set of washes with NaCl/P i . Phagemid particles were eluted using 0.1 M HCl, pH 2.2/1 mgÆmL )1 BSA, neutralized by the addition of 2 M Tris base [26], and either immediately reinfected into E. coli TG1 or stored at 4 °C. Nucleic acid isolation and cloning Following final selection, phagemid particles were infected into E. coli TG1 and propagated as plasmids, followed by DNA extraction. The V NAR cassette was extracted as a NotI/SfiI fragment and subcloned into the similarly restric- ted cloning/expression vector pGC [27]. DNA clones were sequenced on both strands using a BigDye terminator cycle sequencing kit (Applied Biosystems) and a Perkin Elmer Sequenator. The nucleotide sequence of clone 12F-11 is deposited in the GenBank database under accession number AY069988. Recombinant Tom70 protein DNA encoding the receptor domain of Tom70 (residues 111–608) was amplified by PCR, cloned into the vector pET3a as described by Young et al. [21]. For expression, cells were grown to D 600 ¼ 0.6, induced by the addition of isopropyl thio-b- D -galactoside (IPTG, 1 m M final), and recombinant protein purified using Ni-NTA chromatogra- phy (Qiagen) according to the manufacturer’s instructions [21]. Soluble expression of V NAR constructs from expression vector pGC Recombinant proteins were expressed in the bacterial periplasm as described [10]. Briefly, E. coli TG1 starter cultures were grown overnight in 2YT medium containing 100 lgÆmL )1 ampicillin and 2.0% glucose (w/v), diluted 1/100 into fresh 2YT containing 100 lgÆmL )1 ampicillin and 0.1% glucose (w/v) and then grown at 37 °C and shaken at 200 r.p.m. until D 550 ¼ 0.2–0.4. Cultures were then induced with IPTG (1 m M final concentration), grown for a further 16 h at 28 °C and harvested by centrifugation (Beckman JA-14/6K, 5500 g 10 min, 4 °C). Periplasmic fractions were isolated by the method of Minsky [28] and either used as crude fractions or recombinant protein purified by affinity chromatography using an anti-(FLAG) Ig/Sepharose col- umn (10 · 1 cm). The affinity column was equilibrated in NaCl/P i , pH 7.4 and bound protein eluted with Immuno- Pure TM gentle elution buffer (Pierce). Eluted proteins were dialysed against two changes of NaCl/P i containing 0.02% sodium azide, concentrated by ultrafiltration over a 3-kDa cutoff membrane (YM3, Diaflo), and analysed by FPLC on a precalibrated Superdex 200 column (Pharmacia) equili- brated in NaCl/P i buffer pH 7.4. Recombinant proteins were analysed, by SDS/PAGE through 15% Tris/glycine gels. Enzyme linked immunosorbent assays Protein antigens (0.5 lg per well) in NaCl/P i were coated onto Maxisorb Immuno-plates (Nunc) and incubated at 4 °C overnight. Plates were rinsed, blocked with 5% (w/v) Blotto in NaCl/P i for 1 h, and incubated with periplasmic fractions or recombinant protein for 1 h at room tempera- ture. Plates were rinsed with NaCl/P i , washed three times with 0.05% Tween20 in NaCl/P i , and anti-(FLAG) Ig (diluted 1/1000 in 5% Blotto in NaCl/P i ) added. Plates were incubated and washed as above, and the horseradish peroxidase conjugated secondary anti-(mouse Fc) Ig added Table 1. Oligonucleotide primers used in generation of V NAR libraries. Sense primer (fi); antisense primer (‹). Oligonucleotide Number Sequence (5¢ –3¢) Features 5¢ Amplification 8406 (fi) GTCTCGCGGCCCAGCCGGCCATGGCCACAAGGGTAGACCAAACACC N-terminus ¼ TRVDQTP… 5¢ Amplification 8407 (fi) GTCTCGCGGCCCAGCCGGCCATGGCCGCAAGGGTGGACCAAACACC N-terminus ¼ ARVDQTP… 5¢ Amplification 8408 (fi) GTCTCGCGGCCCAGCCGGCCATGGCCGCATGGGTAGACCAAACACC N-terminus ¼ AWVDQTP… 3¢ Amplification 8404 (‹) CACGTTATCTGCGGCCGCTTTCACGGTTAATGCGGTGCC C-terminus ¼ …GTALTVK 3¢ Amplification 8405 (‹) CACGTTATCTGCGGCCGCTTTCACGGTTAATACGGTGCCAGCTCC C-terminus ¼ …GTVLTVK CDR3 Library construction 6981 (‹) GGTTAATACGGTGCCAGCTCCCYYMNNMNNMNNMNNMNNRYHRYH RYHRYHMNNMNNMNNMNNMNNMNNTGCTCCACACTTATACGTGCCACTG 15 residue randomised loop CDR3 Library construction 7211 (‹) TTTCACGGTTAATACGGTGCCAGCTCCTTTCTCMNNMNNMNNMNNRYHR YHRYHRYHRYHMNNMNNMNNMNNMNNGNATGCTCCACACTTATACGT GCC 16 residue randomised loop CDR3 Library construction 6980 (‹) GGTTAATACGGTGCCAGCTCCCYYMNNMNNMNNMNNMNNMNNMNNRYHRY HRYHRYHMNNMNNMNNMNNMNNMNNTGCTTGACACTTATACGTGCC ACTG 17 residue randomised loop CDR3 Library construction 7210 (‹) TTTCACGGTTAATACGGTGCCAGCTCCTTTCTCMNNMNNMNNMNNMNN MNNRYHRYHRYHRYHRYHMNNMNNMNNMNNMNNGNATGCTTGA CACTTATACGTGCC 18 residue randomised loop Ó FEBS 2003 An IgNAR variable domain specific for human Tom70 (Eur. J. Biochem. 270) 3545 [1/1000 in NaCl/P i /5% Blotto]. Plates were washed again and developed using 2,2-azino di-(ethyl) benzthiazoline sulphonic acid (Boehringer Mannheim) and read at A 405 . Biosensor binding analysis A BIAcore TM 1000 biosensor (BIAcore AB, Uppsala, Sweden) was used to measure the interaction between V NAR protein 12F-11 and Tom70. FPLC-purified Tom70 at a concentration of 20 lgÆmL )1 in 10 m M sodium acetate buffer, pH 4.5 was immobilized onto a CM5 sensor chip via amine groups using the amine coupling kit (BIAcore AB) [29]. The immobilization was performed at 25 °Cand 5 lLÆmin )1 flow rate. Injection of 28 lL of Tom70 coupled 990 RU to the surface. Binding experiments were performed in Hepes buffered saline (HBS; 10 m M Hepes, 0.15 M NaCl, 3.4 m M EDTA, 0.005% surfactant P20, pH 7.4) at 25 °C and a constant flow rate of 5 lLÆmin )1 with a series of 12F-11 concentrations (2.2–17.8 n M ). Binding experiments were performed immediately, as prolonged washing with the HBS buffer resulted in a decrease in activity of the immobilized Tom70. Regeneration of the Tom70 surface was achieved by running the dissociation reaction to completion before the next injection of analyte. For binding experiments in the reverse orientation, recombinant protein 12F-11 was immobilized by the standard amine coupling method. 12F-11 protein at a concentration of 20 lgÆmL )1 in 10 m M sodium acetate pH 6.0 was injected for 8 min (40 lL) over an activated surface to couple 650 RU of protein onto the sensor surface. The 12F-11 surface was regenerated with a 10-lL aliquot of 50 m M HCl with negligible loss of binding activity. Binding experiments were performed in HBS buffer at 25 °Canda flow rate of 5 lLÆmin )1 with a range of Tom70 concentra- tions (37.0–296 n M ). The binding data was evaluated with BIAEVALUATION 3.0.2 [30]. Structural modeling V NAR domains 12F-11 (this study) and 7R-1 [10] were modelled using the program MODELLER [31]. The PDB database was searched with the 12F-11 V NAR sequence and 17 structures with the best Z-scores selected as templates. The PDB accession numbers for the template molecules were as follows with the chain Ids indicated: 1B88:B, 1D9K:E, 1H5B:B, 1KB5:A, 1KJ2:A (T-cell receptor alpha domains); 1BJM:A, 2RHE (Bence–Jones proteins); 1AY1:H, 1BGX:H, 1IGF:J, 1KEN:T, 2IGF:H (V H domains); 1IAI:M, 1F6L:L, iIFF:L, 1JNL:L and 35C8:L (V L domains). The resulting model structures were refined and energy minimized using molecular dynamics restrained to the template structure, except where gaps occurred in the alignments and for CDRs 1 and 3. In these cases extensive loop modelling was undertaken and the final model selection based on the modeller objective score. Construction of the 12F-11DCys mutant and 12F-11 reduction/alkylation For reduction and alkylation of 12F-11, recombinant protein (1.3–1.5 mg) was denatured/reduced using 6 M guanidine HCl and 50 m M dithiothreitol (pH 8.0) for 1 h at 45 °C under nitrogen. Cysteine residues were then alkylated by the addition of 100 m M (final) iodo-acetamide (pH 8.0)/1 h (room temperature) followed by quenching with additional dithiothreitol. Samples were dialysed against four changes of NaCl/P i , concentrated, and analysed by FPLC, SDS/PAGE and ELISA, as above. The disulphide minus variant of 12F-11 incorporating mutations Cys22Ala and Cys82Val was constructed by overlapping PCR using oligonucleotide primers N8517 (Forward: 5¢-ACAAGGG TAGACCAAACACCAAGAACAGCAACAAAAGAG ACGGGCGAATCACTGACCATCAACgccGTCCTGA GAGAT-3¢) and N8518 (Reverse: 5¢-TTTCACGGTTAA TGCGGTGCCAGCTCCCCAACTGTAATAAATACC AGACAAATTATATGCTCCaacCCTATACGTGCCA CTG-3¢); followed by secondary PCR using V NAR terminal oligonucleotide primers [17] to complete the framework and incorporate NotIandSfiI restriction endonuclease sites for cloning. Bacterial expression was as described above. Affinity maturation by error-prone PCR The 12F-11 V NAR cassette was mutagenized by error prone PCR using Taq DNA polymerase [32]. Pools of mutated V NAR cassettes were isolated, cut with SfiI/NotI, cloned into the phagemid vector pFAB.5c, and transformed into E. coli TG1 as above. The resulting library ( 1 · 10 6 independent clones) showed on average 1–2 residue changes/100 amino acids. Two rounds of panning under high stringency conditions were performed on immobilized Tom70 as above, except that the final five washes for each selection round incorporated a further 2 min incubation to promote dissociation. The selected V NAR cassettes were then rescued, subcloned and analysed. Results Construction of an expanded Wobbegong V NAR library We previously designed and constructed a Wobbegong IgNAR variable domain (V NAR ) library with long synthetic CDR3 loops of either 15 or 17 residues in length inserted into a mixed scaffold repertoire of 26 naturally occurring V NAR domains. This small library ( 3 · 10 7 independent clones) was displayed on the surface of fd bacteriophage and successfully panned against protein antigens [10]. In order to increase the diversity of possible antigen-binding fragments, this library was expanded in three ways: (a) the extended library comprised increased complexity with CDR3 lengths ranging form 15–18 residues to reflect the predominant natural diversity in Wobbegong and Nurse shark V NAR s (Fig. 1A). Additionally, a different randomization pattern was used, biased toward the incorporation of cysteine at CDR3 loop residue positions 1 and 7–11, to enhance the possibility of inter- and intra CDR disulphide cross-links. These strategies are summarized in Fig. 1B and Table 1, and details of the library construction are given in greater detail in the Materials and methods. (b) The extended library was based on CDR3 loops grafted into a large scaffold repertoire of natural V NAR domains generated by direct RT-PCR from total RNA extracted from Wobbegong shark peripheral blood lymphocytes. Thus, many differing CDR1 sequences and minor framework variations were represented in the 3546 S. D. Nuttall et al. (Eur. J. Biochem. 270) Ó FEBS 2003 extended library. (c) A subset of the extended library now also included naturally occurring CDR3 loops derived directly from the immune repertoire of several sharks. These natural sequences form part of the matured shark immune response, generated in response to exposure to antigen in the natural environment, and have extensive size heterogeneity (as indicated in Fig. 1A) and different randomizations from those used for the synthetic CDR3s [17]. Taken together, these changes resulted in an expanded V NAR library consist- ing of over 4 · 10 8 independent clones, representing signi- ficantly enhanced diversity compared to our original V NAR repertoire. This library was displayed as a fusion with the gene3protein of fd bacteriophage in the vector pFAB5c.HIS (Fig. 1C) allowing for standard phage display and selection. Biopanning against immobilized mitochondrial import receptor Tom70 Tom70 is an integral membrane protein consisting of a large globular receptor domain exposed to the cytosol and a short N-terminal transmembrane anchor. A truncated Tom70 protein construct consisting of residues 111–608 of human Tom70 was expressed in E. coli, purified as a soluble extracellular 60 kDa protein, and immobilized on immuno- tubes as a target protein [21]. The V NAR library was transformed into E. coli TG1 and phagemid particles rescued and panned against the immo- bilized Tom70 antigen. Four rounds of biopanning were performed with an increase in the stringency of washing at each step, and between selection rounds three and four a significant ( 1000-fold) increase in the titre of eluted bacteriophage was observed. Colony PCR on transfected bacteriophage showed that 100% of colonies were positive for V NAR sequences, and this combined with the increase in the titre, indicated positive selection. Thus, V NAR cassettes were rescued from phagemids, cloned into the periplasmic expression vector pGC, and transformed into E. coli TG1. Periplasmic fractions from recombinant clones were tested for binding to Tom70 and negative control antigens by ELISA (not shown). Several clones showed significant binding above background, and all of these proved to be identical sequences. One of these, designated clone 12F-11, was chosen for further analysis. The primary and deduced amino acid sequences of clone 12F-11 are presented in Fig. 2A, including in-frame dual octapeptide FLAG epitope tags and two alanine linker regions. The protein represents a 103 residue V NAR domain, with a predicted molecular mass of 14 054 Da (including the affinity tags). An alignment of protein 12F-11 with four other V NAR proteins showed a high level of sequence conservation except in the CDR regions (Fig. 2B). How- ever, and most surprisingly, the alignment also revealed that the Thr39 residue, present in the other V NAR s, was absent in the 12F-11 protein (Fig. 2B, arrowed). Equally unusual was the CDR3 structure that at 10 residues ( N YN LSGIYYSW C ) is significantly shorter than the 15–18 residues encoded within the library design. This truncation in the CDR3 size probably occurred either during the initial PCR-based library construction, or under selection pressure within the early rounds of panning. The presence of such CDR deletions in large libraries is not uncommon [33], and indeed one V NAR protein we reported previously was an obvious deletion mutant [10]. Recombinant protein 12F-11 is a monomeric, correctly folded protein Loss of a nonCDR residue is potentially destructive to immunoglobulin domain structures. Thus, to determine Fig. 1. Design of the V NAR library. (A) Cumulative frequency histo- gram of IgNAR CDR3 loop lengths, from Wobbegong sharks (26 sequences) and Nurse sharks (35 sequences). (B) Schematic diagram of synthetic CDR3s used in V NAR library construction, showing the randomization patterns used for the varying length CDRs. X repre- sents use of the nucleotide randomization strategy (NNK) that encodes any residue or an amber stop codon. Surrounding framework regions are also shown. (C) V NAR cassettes were ligated into phagemid vector pFAB.5c at the SfiIandNotI restriction endonuclease sites. The phagemid vector incorporates a lacZ promoter, and in-frame PelB leader, Ala 3 linker, and DGene3 protein domains prior to a translation termination codon. Ó FEBS 2003 An IgNAR variable domain specific for human Tom70 (Eur. J. Biochem. 270) 3547 whether the absence of residue Thr39 had an adverse effect upon protein 12F-11 expression, folding, and stability, we undertook a thorough protein chemical analysis. Recom- binant 12F-11 protein was expressed in E. coli and then isolated from the bacterial periplasm by affinity chromato- graphy using an anti-(FLAG) Ig affinity resin. Expression levels obtained were routinely between 2 and 3 mgÆL )1 of protein from shake-flask cultures. Analysis of affinity- isolated protein by FPLC through a precalibrated Superdex 200 gel filtration column showed a single peak eluting from the column at approximately 36 min, corresponding to a protein of 14–15 kDa molecular mass and consistent with the size of a monomeric V NAR domain. (Fig. 3A). There was no evidence of protein aggregation, nor were higher order multimers such as protein dimers or tetramers observed. Indeed, protein 12F-11 appeared to have at least equal stability to other V NAR proteins we have analysed as this FPLC profile was maintained consistently, even after prolonged storage at 4 °C or multiple freeze-thaw cycles, with no evidence of protein degradation. To confirm that the isolated protein was being correctly processed in the E. coli periplasm, N-terminal amino acid sequence analysis on material eluted from the FLAG column showed that only one protein species was present ( 1 TRVDQTP-) corresponding to the predicted N-terminus (Fig. 2A). Far ultraviolet circular dichroism (CD) spectra of aqueous solutions of protein 12F-11 showed a profile with a negative band with k max at 217–219 nm (Fig. 3B). This spectrum is characteristic of a protein with a b-sheet structure with unstructured loops contributed by the CDRs and FLAG affinity tags, and is not a disordered structure [34]. Indeed, the 12F-11 specturm is very similar to CD spectra obtained for other V NAR proteins, for example V NAR 12A-9 that has different CDR loops and a slightly different b-sheet framework, and which is shown for comparison (Fig. 3B) [17]. Together, these results suggest that despite the absence of Thr39, protein 12F-11 folds into compact, b-sheet immunoglobulin in the E. coli periplasm. Indeed, in preliminary structural studies, protein 12F-11 crystallises in the monoclinic P2 space group (results not shown), Fig. 2. Nucleotide and deduced amino acid sequences of the V NAR 12F- 11 variable domain. (A) Nucleotide and deduced amino acid sequences of clone 12F-11. The conserved termini dictated by the oligonucleotide primer sequences used in library construction are underlined, and the alanine linker and dual octapeptide FLAG tags are italicised. The positions of the CDR1 and )3 regions are indicated in bold type. (B) Alignment of protein 12F-11 with four other V NAR domain amino acid sequences (GenBank AY069988; AF336094; AF336087; AF336088; AF336089). Amino acids are designated with the single- letter code, and identical residues (dark shading) and conservative replacements (light shading; I/V/L/M, D/E, K/R, A/G, T/S, Q/N, F/Y) are indicated. The framework residue at position 39, absent in 12F-11, is indicated by the arrow, and the CDR1 and )3 regions are highlighted. Fig. 3. Size exclusion chromatography and CD analysis of protein 12F-11. (A) Elution profile of affinity purified 12F-11 protein on a calibrated Superdex 200 gel-filtration column equilibrated in NaCl/P i , pH 7.4 and run at a flow rate of 0.5 mLÆmin )1 . Protein 12F-11 elutes at approximately 36 min consistent with a monomeric domain (12F-11 calculated M r of 14 054 Da, including linker and dual FLAG octa- peptide tags). Approximate elution times for a series of protein standards are indicated by arrows, and the absorbance at A 214 (unbroken line) and A 280 (dashed line) is given in arbitrary units. The A 214 absorbance peak at approximately 46 min represents sodium azide. The inset shows the same sample analysed by SDS/PAGE through a 15% (w/v) polyacrylamide Tris/glycine gel and stained with Coomassie Brilliant Blue. (B) Circular dichroic spectrum of affinity purified V NAR 12F-11 in 0.05 M sodium phosphate buffer, pH 7.4 (unbroken line). For comparison, the spectrum for the naturally occurring V NAR domain 12A-9 [17] is also shown (dotted line). 3548 S. D. Nuttall et al. (Eur. J. Biochem. 270) Ó FEBS 2003 providing further evidence for folding into an ordered domain structure. To explain more fully why protein 12F-11 folds into a functional protein while missing a framework residue, we modelled the variable domain structure and compared it to a model of a conventional V NAR (Fig. 4). Our modelling studies indicate that Thr39 is located at the end of the C strand, and that the adjacent C-C¢ loop is therefore amenable to structural changes imposed by the residue deletion without disruption to the framework. Otherwise, there is good agreement between the two V NAR models, with the obvious exception of the CDR loop diversity. Specificity and binding activity of recombinant protein 12F-11 The specificity of protein 12F-11 for Tom70 was demon- strated by ELISA (Fig. 5A). Recombinant protein reacted specifically with Tom70 but not several other antigens tested and was concentration dependent to 0.6 pmol of protein (Fig. 5B). The binding kinetics of the 12F)11/Tom70 interaction were also measured by BIAcore biosensor analysis with Tom70 protein immobilized via amine coup- ling to the sensor surface. As immobilized Tom70 on the sensor surface was found to be unstable, with a 70% loss of binding activity in 24 h, binding experiments were per- formed immediately upon immobilization and Fig. 6A shows the interaction of varying concentrations (17.8 n M , 8.9 n M , 4.5 n M , 2.2 n M ) of peak-purified 12F-11 monomer with the immobilized Tom70. Analysis of the binding data with the 1 : 1 Langmuir binding model showed a good fit (Fig. 6A) for the monovalent analyte (12F-11) binding to a Tom70 epitope consistent with a 1 : 1 binding interaction. There was no binding of the 12F-11 monomer to a blank surface (activated and then blocked with ethanolamine) indicating that there is no nonspecific interaction with the sensor surface (Fig. 6A, inset). Kinetic analysis of 12F-11 binding to immobilized Tom70 revealed a rapid association rate constant (k a ¼ 1.68 ± 0.27 · 10 6 M )1 Æs )1 )andadis- sociation rate constant (k D )of3.49±0.36 – 3Æs )1 to yield a K D of 2.2 ± 0.31 )9 M )1 . V NAR protein 12F-11 monomer was also immobilized via amine coupling onto the sensor chip to measure the binding interaction in the reverse orientation. The 12F-11 surface was stable and could be regenerated with 50 m M HCl Fig. 4. Models of V NAR domains. V NAR s 12F-11 (purple framework with magenta CDRs) and 7R-1 (dark green framework with light green CDRs) were modelled on existing immunoglobulin superfamily variable domain structures. The position of residue Thr39, missing in 12F-11, is indicated. Fig. 5. Analysis of protein 12F-11 by ELISA. (A) Protein 12F-11 was purified from the periplasmic fraction of E. coli TG1byaffinity chromatography through an anti-FLAG M2 antibody column and 150 pmol tested for binding to Tom70, lysozyme, kgp (lysine specific gingipain protease from Porphyromonas gingivalis), and a-amylase. Results represent the average of triplicate wells. (B) As for (A) except serial twofold dilutions of protein 12F-11 were tested for binding to Tom70 and lysozyme. Results represent the average of duplicate wells. Ó FEBS 2003 An IgNAR variable domain specific for human Tom70 (Eur. J. Biochem. 270) 3549 without loss of binding activity. The binding data for the interaction of Tom70 to immobilized 12F-11 however, did not fit the theoretical Langmuir model for 1 : 1 binding (Fig. 6B) but displayed biphasic binding characteristics indicative of multivalent binding. This result is consistent with the observation that Tom70 elutes as an equilibrium mixture between dimer (M r  136 kDa) and monomer on size exclusion chromatography. Removal of the 12F-11 V NAR framework disulphide linkage Next, protein 12F-11 was used to test the utility of V NAR domains as possible ÔintrabodiesÕ for expression and use in in vivo targeting applications. Specifically, we asked whether binding affinity was retained in the absence of the conserved immunoglobulin intradomain disulphide bond. In an initial series of experiments, recombinant 12F-11 protein was denatured and reduced using guanidine HCl and dithio- threitol, followed by alkylation of the cysteine residues and refolding. However, in a result seen for many V H /V L antibodies, the modified protein almost exclusively precipi- tated in the soluble fraction. Only a small proportion consistently remained in the soluble fraction, and this protein showed a similar FPLC profile to the unmodified protein (Fig. 7A). This soluble protein retained binding affinity for Tom70 by ELISA (Fig. 1C), and we hypothesize that this fraction represents protein that was not fully alkylated and was thus able to refold, with probable reoxidation of the disulphide bond. In contrast, the insoluble fraction most likely represents irreversibly aggre- gated alkylated material. In order to more systematically test this hypothesis, we elected to eliminate the possibility of disulphide bond formation genetically by replacement of residues Cys22 and Cys82 with alanine and valine, respectively, to give a cysteine minus mutant (12F-11DCys). Use of alanine and valine was initially determined in a set of competitive selection experiments [35], and replacement with this pair of residues maintains a hydrophobic character suitable for amino acids buried deep within the protein interior and closely approximates the relative size of the cysteine side chains. When 12F-11DCys was expressed into the E. coli periplasm, expression levels comparable to those obtained for the wild-type were obtained, and both wild-type and mutant proteins were indistinguishable by gel filtration chromatography (Fig. 7B). However, when binding to Tom70 was assessed by ELISA under stringent washing conditions, 12F-11DCys did not appear to target Tom70 compared to 12F-11 and the refolded 12F-11 (reduced/alkylated soluble fraction) proteins (Fig. 7C). This surprising result was further tested by kinetic ana- lysis by biosensor, which showed an approximately 200-fold decrease in binding affinity (K D ¼ 2n M to K D ¼500 n M ) (Fig. 7D). Interestingly, this loss in bind- ing affinity was completely attributed to a slower association phase, with the dissociation curve indistinguishable from the parent protein. We suggest that removal of the stabilizing rigidity provided by the disulphide bond results in a slight perturbation of the orientation of the immunoglobulin b-sheets relative to each other. However, upon binding, the interaction with antigen functions to lock the b-sheet and CDR loop conformations, resulting in the original dissoci- ation kinetics. Affinity maturation of 12F-11 In order to affinity mature V NAR 12F-11, a library of mutant proteins was generated by error-prone PCR. The resultant bacteriophage-displayed library ( 10 6 independ- ent clones) was panned against Tom70 under conditions designed to select for variants with enhanced off-rate kinetics. After two rounds of selection a >4000-fold increase in titre was observed indicating strong selection. Of the resultant clones, a large proportion (64%) were 12F- 11 wild-type, while those with mutations almost exclusively showed conservative framework variations well-removed from the V NAR binding site. Only one variant, designated 15Z-2, showed changes mapping to either the CDR or Fig. 6. Analysis of protein 12F-11 by BIAcore. (A) Binding of mono- meric V NAR protein 12F-11 to immobilized Tom70 protein (990 RU) was measured at a constant flow rate of 5 lLÆmin )1 with an injection volume of 35 lL. Dissociation was continued with HBS buffer until the response returned to the initial value before injecting the next sample. The inset shows the binding profile of protein 12F-11 (20 lgÆmL )1 ) to immobilized Tom70 and a blank surface (NHS/EDC activated and blocked with ethanolamine). The circles show the fit to the data obtained on analysis with the 1 : 1 Langmuir binding model for evaluation of the kinetic rate constants. (B) Sensorgram showing the binding of Tom70 (74 n M ) to immobilized V NAR protein 12F-11 monomer (650 RU) at a constant flow rate of 5 lLÆmin )1 .Thecircles show the fit to the data on analysis with the 1 : 1 Langmuir binding model when only the first 110 s of the dissociation phase is analysed. 3550 S. D. Nuttall et al. (Eur. J. Biochem. 270) Ó FEBS 2003 CDR-framework junctions. The two mutations in this clone were Lys33 fi Arg, which is in the CDR1 loop (Fig. 1A), and Thr43 fi Ile, which is a conservative framework variant. Protein 15Z-2 expressed at levels similar to the parental type and exhibited similar behaviour upon FPLC analysis (Fig. 8A). When tested by ELISA, 15Z-2 showed a slightly higher binding response to Tom70, but not negative control antigens (Fig. 8B). However, this difference was not apparent in kinetic measurements by biosensor, where 12F-11 and 15Z-2 showed indistinguishable binding kinet- ics, including no differences in dissociation rates. Moreover, upon extended storage, protein 15Z-2 showed a slight tendency to precipitation, suggesting that its coselection with wild-type, and slightly higher ELISA responses, may be attributable to an increased tendency toward aggregation. Discussion The aim of this study was to generate an in vitro library of V NAR domains, containing both synthetic and natural CDR3 loops, and then to isolate specific binding molecules using as an initial target antigen the mitochondrial outer membrane receptor Tom70. The resulting protein, 12F-11, shows a high degree of affinity and specificity for Tom70, andwithaK D of  2n M compares very well with affinities reported for camelid V H H domains (that vary in the range 2–300 n M [36]) and for scFv and disulphide stabilized Fv fragments [37]. The high affinity of 12F-11 is directly attributable to a relatively rapid association rate (k a  1.7 · 10 6 M )1 Æs )1 ), while the dissociation rate (k Da  3.5 · 10 )3 Æs )1 ) is more typical of other V NAR and camelid V H H single domain antibodies [36]. For example, previously we described a naturally occurring V NAR targeting the kgp protease from Porphyromonas gingivalis, and there the K D was 1.31 · 10 )7 M , primarily due to the lower association rate (k a ¼ 4.3 · 10 4 M )1 Æs )1 ) [17]. Structurally, protein 12F-11 is a slightly unusual member of the V NAR family. Firstly, the absence of a framework residue which is commonly present (Thr39), could be hypothesized to deform the underlying b-framework. However, the data clearly demonstrates that this is not the case, and molecular modelling maps this residue to a region distant to the antigen-binding site, and on the periphery of the immunoglobulin-like core scaffold and adjacent to the C-C¢ loop (see Fig. 4). Further, there may well be even greater latitude for mutations within this region, as in unrelated experiments we have also discovered a naturally occurring V NAR lacking five residues in this loop position (S. Nuttall, unpublished data). Secondly, the mean size of the V NAR CDR3 loop is 16 residues, yet protein 12F-11 achieves low nanomolar affinity binding with a CDR3 only 10 residues in length. While this apparently contradicts the Fig. 7. Effect of removal of the 12F-11 disulphide bond. (A) Elution profiles of affinity purified 12F-11 protein (dotted line), and the soluble fraction remaining after reduction and alkylation (unbroken line), on a calibrated Superose 12 gel-filtration column equilibrated in NaCl/P i , pH 7.4 and run at a flow rate of 0.5 mLÆmin )1 . The inset shows relative amounts of material recovered from the soluble (S) and insoluble (P) fractions after reduction and alkylation compared to untreated protein (U). (B) Elution profiles of affinity purified 12F-11 (unbroken line) and 12F-11DCys (dotted line) proteins analysed as in (A). (C) ELISA comparing binding of 12F-11, 12F-11 reduced and alkylated soluble fraction, and 12F-11DCys proteins to Tom70 and negative control antigens. Results represent the average of duplicate wells. (D) Binding of monomeric proteins 12F-11 and 12F-11DCys (40 lgÆmL )1 ) to immobilized Tom70 protein. The inset shows the binding profile of protein 12F-11DCys to immobilized Tom70 and to a nonspecific antigen and a blank surface (NHS/EDC activated and blocked with ethanolamine). Ó FEBS 2003 An IgNAR variable domain specific for human Tom70 (Eur. J. Biochem. 270) 3551 theory that V NAR s encompass most of their binding affinity within a long and diverse CDR3 loop, it is significant that some naturally occurring V NAR s, presumably selected by the shark antigen-driven immune response, have even shorter CDR3 lengths [10]. Indeed, not all antibody V-like domains display extended CDR3 loops, by analogy to a recent structural analysis of camelid V H H domains [38] a significant proportion of the V NAR repertoire may comprise CDR3 loops overlayed onto and across the scaffold surface. This conclusion means that, in the context of antigen binding surfaces, shark V NAR and camelid V H H libraries will contain structural homologues similar to antibody V H and V L libraries, as well as providing discrete and distinctly different structural repertoires. In an attempt to further improve the 12F-11 binding affinity, we generated a library of affinity-matured variants. However, selection failed to isolate any mutants with improved binding kinetics and instead there was strong selection for the wild-type protein. This probably reflects a precise interaction between the CDR1 and relatively short CDR3 loop with only minor variations being tolerated. Thus, within the relatively limited context of a library of a million independent clones covering the entire V NAR cassette, it is unlikely that the rare beneficial mutations will be present. In contrast, in experiments aimed at affinity maturing other V NAR domains, we have isolated several variants with approaching order-of-magnitude enhanced affinities from similar sized error-prone PCR libraries. However, in these cases the mutations targeted an extended and flexible CDR3 loop, which provided greater latitude for the introduction of positive mutations (S. Nuttall, unpub- lished observation). The experiments testing the impact of removal of the intradomain disulphide bond provide further evidence that V NAR 12F-11 is a tightly folded protein domain relatively intolerant to manipulation. Reduction and alkylation most probably had a fatal impact upon protein stability and folding resulting in aggregated protein. This is not uncom- mon for immunoglobulin domains, and only relatively few antibody V H or V L domains remain functional after such modification [39–41]. This is unsurprising given that during alkylation, cysteine is replaced by S-carboxymethylate cysteine, resulting in a significant increase in molecular mass that must be accommodated within the otherwise tightly folded antibody core. More elegant is genetic replacement of the two half-cystines with an alanine-valine pair that is virtually mass neutral and represents an optimum replacement strategy for antibodies determined in a competitive selection system [35]. Our finding that such a substitution, with consequent ablation of the intramole- cular disulphide bond, decreases the kinetics of association but not dissociation can be explained as due to a slight perturbation of the orientation of the immunoglobulin b-sheets relative to each other thereby reducing the associ- ation rate. In contrast, antigen binding presumably locks the b-sheet and CDR loop conformation, resulting in the original dissociation kinetics. This interpretation is consis- tent with the current dogma that antigen binding is critically dependant upon the precise interaction of CDR1, CDR3, and underlying framework residues. Specifically, we noted that the three CDR3 tyrosine residues in 12F-11 (Tyr residues are over-represented in CDR loop structures [42]), may combine with Tyr35 which lies just C-terminal to the CDR1 loop, to form the predominant antigen binding site. Additionally, the noncanonical disulphide bridge often found in V NAR domains, that links the CDR1 and )3 loops together providing additional conformational stabi- lity, is absent in this case. However, any more detailed analysis of the 12F-11 paratope and assessment of the varying contributions of CDR and framework regions clearly requires definitive structural data; this analysis is currently in progress. The isolation of 12F-11 from the extended V NAR library demonstrates that synthetic CDR3 libraries selected in vitro can generate proteins with antigen-binding affinities at least equal to those of natural systems (i.e. immunization of animals followed by isolation of the variable gene reper- toire). This is especially important where conventional (i.e. murine) antibodies are difficult to generate. The human Tom70 receptor is an important example of such refractory targets, and the generation of a high-affinity reagent specific for the cytosolic domain is a potentially valuable tool in the study of preprotein targeting. However, in a series of in vitro import inhibition experiments, designed to test the ability of 12F-11 protein to block the import of precursor proteins Fig. 8. Affinity maturation of 12F-11. (A) Elution profiles of affinity purified 12F-11 (dotted line) and 15Z-2 (unbroken line) proteins on a calibrated Superose 12 gel-filtration column equilibrated in NaCl/P i , pH 7.4 and run at a flow rate of 0.5 mLÆmin )1 . (B) ELISA comparing binding of 12F-11 and 15Z-2 protein to Tom70 and negative control antigens. Results represent the average of duplicate wells. 3552 S. D. Nuttall et al. (Eur. J. Biochem. 270) Ó FEBS 2003 [...]...Ó FEBS 2003 An IgNAR variable domain specific for human Tom70 (Eur J Biochem 270) 3553 into rat liver mitochondria, no inhibition was observed, suggesting that 12F-11 binds an epitope of the Tom70 soluble domain that is inaccessible in the endogenous form, for example through oligomerization [43], or by it facing the membrane This, combined with the lowered affinity of the 12F-11DCys variant, raises... 30 kDa for the dimer, equivalent to one scFv) making them attractive reagents for future diagnostic and possibly therapeutic purposes [49] Acknowledgements We thank Drs Alex Kortt, Lindsay Sparrow, Jacqui Gulbis and Mike Gorman, and Ms Chaille Webb for advice and discussions We are indebted to Mr Andreas Fischer for his assistance with collection of blood samples from Wobbegong sharks We thank Dr Terry... mitochondria of mammalian cells Biochim Biophys Acta 1592, 97–105 19 Suzuki, H., Maeda, M & Mihara, K (2002) Characterization of rat TOM70 as a receptor of the preprotein translocase of the mitochondrial outer membrane J Cell Sci 115, 1895–1905 20 Brix, J., Dietmeier, K & Pfanner, N (1997) Differential recognition of preproteins by the purified cytosolic domains of the mitochondrial import receptors, Tom20,... raises doubts as to the utility of these particular VNARs as intracellular targeting reagents The VNAR library described here is of sufficient size and diversity to be an important resource, particularly for screening against large proteinaceous antigens Future advances likely to expand these applications include improvements in initial panning and screening, followed by the application of more sophisticated... Hudson, P.J (2001) Isolation of the new antigen receptor from Wobbegong sharks, and use as a scaffold for the display of protein loop libraries Mol Immunol 38, 313–326 11 Roux, K.H., Greenberg, A.S., Greene, L., Strelets, L., Avila, D., McKinney, E.C & Flajnik, M.F (1998) Structural analysis of the nurse shark (new) antigen receptor (NAR): molecular convergence of NAR and unusual mammalian immunoglobulins... hypermutation of the new antigen receptor gene (NAR) in the nurse shark does not generate the repertoire: possible role in antigen-driven reactions in the absence of germinal centers Proc Natl Acad Sci USA 95, 14343–14348 16 Diaz, M., Velez, J., Singh, M., Cerny, J & Flajnik, M.F (1999) Mutational pattern of the nurse shark antigen receptor gene (NAR) is similar to that of mammalian Ig genes and to spontaneous... Mulhern for performing the CD spectral analysis and Mr N Bartone for oligonucleotide synthesis, and Mr P Strike for assistance with biosensor measurements References 1 Chothia, C., Lesk, A.M., Tramontano, A., Levitt, M., Smith-Gill, S.J., Air, G., Sheriff, S., Padlan, E.A., Davies, D., Tulip, W.R., Colman, P.M., Spinelli, S., Alzari, P.M & Poljak, R.J (1989) Conformations of immunoglobulin hypervariable... Determination of relative binding affinity of influenza virus N9 sialidases with the Fab fragment of monoclonal antibody NC41 using biosensor technology Eur J Biochem 217, 319–325 30 Kortt, A.A., Nice, E & Gruen, L.C (1999) Analysis of the binding of the Fab fragment of monoclonal antibody NC10 to influenza virus N9 neuraminidase from tern and whale using the BIAcore biosensor: effect of immobilization level and. .. 25 Engberg, J., Andersen, P.S., Nielsen, L.F., Dziegiel, M., Johansen, L.K & Albrechtsen, B (1996) Phage-display libraries of murine and human antibody Fab fragments Molec Biotechnol 6, 287– 310 26 Galanis, M., Irving, R.A & Hudson, P.J (1997) Bacteriophage library construction and selection of recombinant antibodies In Current Protocols in Immunology, pp 17.1.1–17.1.45 John Wiley and Sons, New York... new antigen receptor gene family that undergoes rearrangement and extensive somatic diversification in sharks Nature 374, 168–173 6 Nuttall, S.D., Irving, R.A & Hudson, P.J (2000) Immunoglobulin VH domains and beyond: design and selection of singledomain binding and targeting reagents Current Pharmaceut Biotechnol 1, 253–263 7 Diaz, M., Stanfield, R.L., Greenberg, A.S & Flajnik, M.F (2002) Structural analysis, . Isolation and characterization of an IgNAR variable domain specific for the human mitochondrial translocase receptor Tom70 Stewart D. Nuttall 1,2 ,. Stewart.Nuttall@csiro.au Abbreviations: IgNAR, new antigen receptor antibody from sharks; VNAR, single variable domain of the IgNAR antibody; Tom70, human 70 kDa component of the translocase

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