Refined crystal structure of the influenza virus N9 neuraminidase-NC41 Fab complex.

The crystal structure of the complex between neuraminidase from influenza virus (subtype N9 and isolated from an avian source) and the antigen-binding fragment (Fab) of monoclonal antibody NC41 has been refined by both least-squares and simulated annealing methods to an R-factor of 0.191 using 31,846 diffraction data in the resolution range 8.0 to 2.5 A. The resulting model has a root-mean-square deviation from ideal bond-length of 0.016 A. One fourth of the tetrameric complex comprises the crystallographic model, which has 6577 non-hydrogen atoms and consists of 389 protein residues and eight carbohydrate residues in the neuraminidase, 214 residues in the Fab light chain, and 221 residues in the heavy chain. One putative Ca ion buried in the neuraminidase, and 73 water molecules, are also included. A remarkable shape complementarity exists between the interacting surfaces of the antigen and the antibody, although the packing density of atoms at the interface is somewhat looser than in the interior of a protein. Similarly, there is a high degree of chemical complementarity between the antigen and antibody, mediated by one buried salt-link, two solvated salt-links and 12 hydrogen bonds. The antibody-binding site on neuraminidase is discontinuous and comprises five chain segments and 19 residues in contact, whilst 33 neuraminidase residues in eight segments have 899 A2 of surface area buried by the interaction (to a 1.7 A probe), including two hexose units. Seventeen residues in NC41 Fab lying in five of the six complementarity determining regions (CDRs) make contact with the neuraminidase and 36 antibody residues in seven segments have 916 A2 of buried surface area. The interface is more extensive than those of the three lysozyme-Fab complexes whose crystal structures have been determined, as judged by buried surface area and numbers of contact residues. There are only small differences (less than 1.5 A) between the complexed and uncomplexed neuraminidase structures and, at this resolution and accuracy, those differences are not unequivocal. The main-chain conformations of five of the CDRs follow the predicted canonical structures. The interface between the variable domains of the light and heavy chains is not as extensive as in other Fabs, due to less CDR-CDR interaction in NC41. The first CDR on the NC41 Fab light chain is positioned so that it could sterically hinder the approach of small as well as large substrates to the neuraminidase active-site pocket, suggesting a possible mechanism for the observed inhibition of enzyme activity by the antibody.(ABSTRACT TRUNCATED AT 400 WORDS)     

A single-domain antibody fragment in complex with RNase A: non-canonical loop structures and nanomolar affinity using two CDR loops

BACKGROUND: Camelid serum contains a large fraction of functional heavy-chain antibodies - homodimers of heavy chains without light chains. The variable domains of these heavy-chain antibodies (VHH) have a long complementarity determining region 3 (CDR3) loop that compensates for the absence of the antigen-binding loops of the variable light chains (VL). In the case of the VHH fragment cAb-Lys3, part of the 24 amino acid long CDR3 loop protrudes from the antigen-binding surface and inserts into the active-site cleft of its antigen, rendering cAb-Lys3 a competitive enzyme inhibitor. RESULTS: A dromedary VHH with specificity for bovine RNase A, cAb-RN05, has a short CDR3 loop of 12 amino acids and is not a competitive enzyme inhibitor. The structure of the cAb-RN05-RNase A complex has been solved at 2.8 A. The VHH scaffold architecture is close to that of a human VH (variable heavy chain). The structure of the antigen-binding hypervariable 1 loop (H1) of both cAb-RN05 and cAb-Lys3 differ from the known canonical structures; in addition these H1 loops resemble each other. The CDR3 provides an antigen-binding surface and shields the face of the domain that interacts with VL in conventional antibodies. CONCLUSIONS: VHHs adopt the common immunoglobulin fold of variable domains, but the antigen-binding loops deviate from the predicted canonical structure. We define a new canonical structure for the H1 loop of immunoglobulins, with cAb-RN05 and cAb-Lys3 as reference structures. This new loop structure might also occur in human or mouse VH domains. Surprisingly, only two loops are involved in antigen recognition; the CDR2 does not participate. Nevertheless, the antigen binding occurs with nanomolar affinities because of a preferential usage of mainchain atoms for antigen interaction.     

Crystal structure of an anti-interleukin-2 monoclonal antibody Fab complexed with an antigenic nonapeptide

The three-dimensional structure of the Fab fragment of a monoclonal antibody (LNKB-2) to human interleukin-2 (IL-2) complexed with a synthetic antigenic nonapeptide, Ac-Lys-Pro-Leu-Glu-Glu-Val-Leu-Asn-Leu-OMe, has been determined at 3.0 A resolution. In the structure, four out of the six hypervariable loops of the Fab (complementarity determining regions [CDRs] L1, H1, H2, and H3) are involved in peptide association through hydrogen bonding, salt bridge formation, and hydrophobic interactions. The Tyr residues in the Fab antigen binding site play a major role in antigen-antibody recognition. The structures of the complexed and uncomplexed Fab were compared. In the antigen binding site the CDR-L1 loop of the antibody shows the largest structural changes upon peptide binding. The peptide adopts a mostly alpha-helical conformation similar to that in the epitope fragment 64-72 of the IL-2 antigen. The side chains of residues Leu 66, Val 69, and Leu 70, which are shielded internally in the IL-2 structure, are involved in interactions with the Fab in the complex studied. This indicates that antibody-antigen complexation involves a significant rearrangement of the epitope-containing region of the IL-2 with retention of the alpha-helical character of the epitope fragment.     

Complex between Peptostreptococcus magnus protein L and a human antibody reveals structural convergence in the interaction modes of Fab binding proteins

BACKGROUND: Peptostreptococcus magnus protein L (PpL) is a multidomain, bacterial surface protein whose presence correlates with virulence. It consists of up to five homologous immunoglobulin binding domains that interact with the variable (VL) regions of kappa light chains found on two thirds of mammalian antibodies. RESULTS: We refined the crystal structure of the complex between a human antibody Fab fragment (2A2) and a single PpL domain (61 residues) to 2.7 A. The asymmetric unit contains two Fab molecules sandwiching a single PpL domain, which contacts similar VL framework regions of two light chains via independent interfaces. The residues contacted on VL are remote from the hypervariable loops. One PpL-Vkappa interface agrees with previous biochemical data, while the second is novel. Site-directed mutagenesis and analytical-centrifugation studies suggest that the two PpL binding sites have markedly different affinities for VL. The PpL residues in both interactions are well conserved among different Peptostreptococcus magnus strains. The Fab contact positions identified in the complex explain the high specificity of PpL for antibodies with kappa rather than lambda chains. CONCLUSIONS: The PpL-Fab complex shows the first interaction of a bacterial virulence factor with a Fab light chain outside the conventional combining site. Structural comparison with two other bacterial proteins interacting with the Fab heavy chain shows that PpL, structurally homologous to streptococcal SpG domains, shares with the latter a similar binding mode. These two bacterial surface proteins interact with their respective immunoglobulin regions through a similar beta zipper interaction.     

Generation and Characterization of High Affinity Humanized Fab Against Hepatitis B Surface Antigen

5S is a mouse monoclonal IgG1 that binds to the ‘a’ epitope of the Hepatitis B surface antigen (HBsAg) and tested positive in an in vitro test for virus neutralization. We have earlier reported the generation of humanized single chain variable fragment (scFv) from the same. In this article we report the generation of a recombinant Fab molecule by fusing humanized variable domains of 5S with the constant domains of human IgG1. The humanized Fab expressed in E. coli and subsequently purified, retained a high binding affinity (K_D = 3.63 nmol/L) to HBsAg and bound to the same epitope of HBsAg as the parent molecule. The humanized Fab also maintained antigen binding in the presence of various destabilizing agents like 3 M NaCl, 30% DMSO, 8 M urea, and extreme pH. This high affinity humanized Fab provides a basis for the development of therapeutic molecules that can be safely utilized for the prophylaxis and treatment for Hepatitis B infection.     

Mutual conformational adaptations in antigen and antibody upon complex formation between an Fab and HIV-1 capsid protein p24

BACKGROUND: Elucidating the structural basis of antigen-antibody recognition ideally requires a structural comparison of free and complexed components. To this end we have studied a mouse monoclonal antibody, denoted 13B5, raised against p24, the capsid protein of HIV-1. We have previously described the first crystal structure of intact p24 as visualized in the Fab13B5-p24 complex. Here we report the structure of the uncomplexed Fab13B5 at 1.8 A resolution and analyze the Fab-p24 interface and the conformational changes occurring upon complex formation. RESULTS: Fab13B5 recognizes a nearly continuous epitope comprising a helix-turn-helix motif in the C-terminal domain of p24. Only 4 complementarity-determining regions (CDRs) are in contact with p24 with most interactions being by the heavy chain. Comparison of the free and complexed Fab reveals that structural changes upon binding are localized to a few side chains of CDR-H1 and -H2 but involve a larger, concerted displacement of CDR-H3. Antigen binding is also associated with an 8 degrees relative rotation of the heavy and light chain variable regions. In p24, small conformational changes localized to the turn between the two helices comprising the epitope result from Fab binding. CONCLUSIONS: The relatively small area of contact between Fab13B5 and p24 may be related to the fact that the epitope is a continuous peptide rather than a more complex protein surface and correlates with a relatively low affinity of antigen and antibody. Despite this, a significant quaternary structural change occurs in the Fab upon complex formation, with additional smaller adaptations of both antigen and antibody.     

Molecular interactions between a recombinant IgE antibody and the beta-lactoglobulin allergen

Allergies are caused by the immune reaction to commonly harmless proteins, allergens. This reaction is typified by immunoglobulin E (IgE) antibodies. We report the crystal structure of an IgE Fab fragment in complex with beta-lactoglobulin (BLG), one of the major allergens of bovine milk. The solved structure shows how two IgE/Fab molecules bind the dimeric BLG. The epitope of BLG consists of six different short fragments of the polypeptide chain, which are located especially in the beta strands, covering a flat area on the allergen surface. All six CDR (complementary-determining region) loops of the IgE Fab participate in the binding of BLG. The light chain CDR loops are responsible for the binding of the flat beta sheet region of BLG. The IgE epitope is different from common IgG epitopes that are normally located in the exposed loop regions of antigens and observed also in the two recently determined allergen-IgG complexes.     

His‐tag binding by antibody C706 mimics β‐amyloid recognition

Alzheimer's disease is a progressive neurodegenerative disease characterized by extracellular deposits of β‐amyloid (Aβ) plaques. Aggregation of the Aβ₄₂ peptide leading to plaque formation is believed to play a central role in Alzheimer's disease pathogenesis. Anti‐Aβ monoclonal antibodies can reduce amyloid plaques and could possibly be used for immunotherapy. We have developed a monoclonal antibody C706, which recognizes the human Aβ peptide. Here we report the crystal structure of the antibody Fab fragment at 1.7 Å resolution. The structure was determined in two crystal forms, P2₁ and C2. Although the Fab was crystallized in the presence of Aβ₁₆, no peptide was observed in the crystals. The antigen‐binding site is blocked by the hexahistidine tag of another Fab molecule in both crystal forms. The poly‐His peptide in an extended conformation occupies a crevice between the light and heavy chains of the variable domain. Two consecutive histidines (His4–His5) stack against tryptophan residues in the central pocket of the antigen‐binding surface. In addition, they form hydrogen bonds to the acidic residues at the bottom of the pocket. The mode of his‐tag binding by C706 resembles the Aβ recognition by antibodies PFA1 and WO2. All three antibodies recognize the same immunodominant B‐cell epitope of Aβ. By similarity, residues Phe–Arg–His of Aβ would be a major portion of the C706 epitope. Copyright © 2010 John Wiley & Sons, Ltd.     

The association of heavy and light chain variable domains in antibodies: implications for antigen specificity

The antigen-binding site of immunoglobulins is formed by six regions, three from the light and three from the heavy chain variable domains, which, on association of the two chains, form the conventional antigen-binding site of the antibody. The mode of interaction between the heavy and light chain variable domains affects the relative position of the antigen-binding loops and therefore has an effect on the overall conformation of the binding site. In this article, we analyze the structure of the interface between the heavy and light chain variable domains and show that there are essentially two different modes for their interaction that can be identified by the presence of key amino acids in specific positions of the antibody sequences. We also show that the different packing modes are related to the type of recognized antigen.     

Structure of the malaria antigen AMA1 in complex with a growth-inhibitory antibody

Identifying functionally critical regions of the malaria antigen AMA1 (apical membrane antigen 1) is necessary to understand the significance of the polymorphisms within this antigen for vaccine development. The crystal structure of AMA1 in complex with the Fab fragment of inhibitory monoclonal antibody 1F9 reveals that 1F9 binds to the AMA1 solvent-exposed hydrophobic trough, confirming its importance. 1F9 uses the heavy and light chain complementarity-determining regions (CDRs) to wrap around the polymorphic loops adjacent to the trough, but uses a ridge of framework residues to bind to the hydrophobic trough. The resulting 1F9-AMA1-combined buried surface of 2,470 A(2) is considerably larger than previously reported Fab-antigen interfaces. Mutations of polymorphic AMA1 residues within the 1F9 epitope disrupt 1F9 binding and dramatically reduce the binding of affinity-purified human antibodies. Moreover, 1F9 binding to AMA1 is competed by naturally acquired human antibodies, confirming that the 1F9 epitope is a frequent target of immunological attack.     

Three-dimensional structures of the free and antigen-bound Fab from monoclonal antilysozyme antibody HyHEL-63(,)

Antigen-antibody complexes provide useful models for studying the structure and energetics of protein-protein interactions. We report the cloning, bacterial expression, and crystallization of the antigen-binding fragment (Fab) of the anti-hen egg white lysozyme (HEL) antibody HyHEL-63 in both free and antigen-bound forms. The three-dimensional structure of Fab HyHEL-63 complexed with HEL was determined to 2.0 A resolution, while the structure of the unbound antibody was determined in two crystal forms, to 1.8 and 2.1 A resolution. In the complex, 19 HyHEL-63 residues from all six complementarity-determining regions (CDRs) of the antibody contact 21 HEL residues from three discontinuous polypeptide segments of the antigen. The interface also includes 11 bound water molecules, 3 of which are completely buried in the complex. Comparison of the structures of free and bound Fab HyHEL-63 reveals that several of the ordered water molecules in the free antibody-combining site are retained and that additional waters are added upon complex formation. The interface waters serve to increase shape and chemical complementarity by filling cavities between the interacting surfaces and by contributing to the hydrogen bonding network linking the antigen and antibody. Complementarity is further enhanced by small (<3 A) movements in the polypeptide backbones of certain antibody CDR loops, by rearrangements of side chains in the interface, and by a slight shift in the relative orientation of the V(L) and V(H) domains. The combining site residues of complexed Fab HyHEL-63 exhibit reduced temperature factors compared with those of the free Fab, suggesting a loss in conformational entropy upon binding. To probe the relative contribution of individual antigen residues to complex stabilization, single alanine substitutions were introduced in the epitope of HEL recognized by HyHEL-63, and their effects on antibody affinity were measured using surface plasmon resonance. In agreement with the crystal structure, HEL residues at the center of the interface that are buried in the complex contribute most to the binding energetics (DeltaG(mutant) - DeltaG(wild type) > 3.0 kcal/mol), whereas the apparent contributions of solvent-accessible residues at the periphery are much less pronounced (<1.5 kcal/mol). In the latter case, the mutations may be partially compensated by local rearrangements in solvent structure that help preserve shape complementarity and the interface hydrogen bonding network.     

Recognition of Mesothelin by the Therapeutic Antibody MORAb-009: STRUCTURAL AND MECHANISTIC INSIGHTS*

Mesothelin is a tumor differentiation antigen that is highly expressed in many epithelial cancers, with limited expression in normal human tissues. Binding of mesothelin on normal mesothelial cells lining the pleura or peritoneum to the tumor-associated cancer antigen 125 (CA-125) can lead to heterotypic cell adhesion and tumor metastasis within the pleural and peritoneal cavities. This binding can be prevented by MORAb-009, a humanized monoclonal antibody against mesothelin currently under clinical trials. We show here that MORAb-009 recognizes a non-linear epitope that is contained in the first 64-residue fragment of the mesothelin. We further demonstrate that the recognition is independent of glycosylation state of the protein but sensitive to the loss of a disulfide bond linking residues Cys-7 and Cys-31. The crystal structure of the complex between the mesothelin N-terminal fragment and Fab of MORAb-009 at 2.6 Å resolution reveals an epitope encompassing multiple secondary structural elements of the mesothelin, including residues from helix α1, the loops linking helices α1 and α2, and between helices α4 and α5. The mesothelin fragment has a compact, right-handed superhelix structure consisting of five short helices and connecting loops. A residue essential for complex formation has been identified as Phe-22, which projects its side chain into a hydrophobic niche formed on the antibody recognition surface upon antigen-antibody contact. The overlapping binding footprints of both the monoclonal antibody and the cancer antigen CA-125 explains the therapeutic effect and provides a basis for further antibody improvement.     

The three-dimensional structure of a T-cell antigen receptor V alpha V beta heterodimer reveals a novel arrangement of the V beta domain.

The crystal structure of a mouse T-cell antigen receptor (TCR) Fv fragment complexed to the Fab fragment of a specific anti-clonotypic antibody has been determined to 2.6 A resolution. The polypeptide backbone of the TCR V alpha domain is very similar to those of other crystallographically determined V alphas, whereas the V beta structure is so far unique among TCR V beta domains in that it displays a switch of the c" strand from the inner to the outer beta-sheet. The beta chain variable region of this TCR antigen-binding site is characterized by a rather elongated third complementarity-determining region (CDR3beta) that packs tightly against the CDR3 loop of the alpha chain, without leaving any intervening hydrophobic pocket. Thus, the conformation of the CDR loops with the highest potential diversity distinguishes the structure of this TCR antigen-binding site from those for which crystallographic data are available. On the basis of all these results, we infer that a significant conformational change of the CDR3beta loop found in our TCR is required for binding to its cognate peptide-MHC ligand.     

Production of recombinant humanized anti-HBsAg Fab fragment from Pichia pastoris by fermentation

In this report, we describe the high-yield secretory expression of the recombinant human anti-HBsAg Fab fragment from Pichia pastoris that was achieved by co-integration of the genes encoding the heavy and light chains (both under the control of alcohol oxidase promoter) into the genome of the yeast cells. The fed-batch fermentations were carried out in a 5 L scale. Both chains of the Fab were successfully expressed upon methanol induction. The absorbance (OD600) of the broth can reach 350 approximately 500 at the end of fed-batch phase. After the induction, the expression level of the recombinant Fab (soluble) reached 420 approximately 458 mg/L. The recombinant Fab fragment was purified from the crude culture supernatant by ion exchange chromatography and the purity of the recombinant Fab fragment was over 95%. The affinity activities of the crude fermentation supernatant and the purified Fab were analyzed by indirect ELISA, which showed that the purified recombinant Fab fragment had high affinity activity with hepatitis B surface antigen.     

Identification of critical contact residues in the NC41 epitope of a subtype N9 influenza virus neuraminidase

We have examined amino acids on influenza virus neuraminidase (NA) subtype N9 (A/tern/Australia/G70c/75) which are in contact with monoclonal antibody NC41 to analyze individual interactions important for antibody recognition. The crystal structure of NA complexed with NC41 Fab¹ shows antibody contacts at 19 amino acid residues on the NA surface which are localized on five polypeptide loops surrounding the enzyme active site. Fifteen mutant NA genes were constructed to encode a protein which contained a single amino acid substitution and these were tested for effects of the replacement on NC41 binding. Our data revealed that NAs with changes at 368, 400, and 434 completely lost NC41 recognition. NAs with side chains replaced at residues 346 and 373 exhibited binding reduced to less than 50% of wild-type binding. Changes in seven other contacting residues, including substituted side chains which differed considerably from wild-type NA in size and charge, had no significant effect on NC41 binding. These results indicate that only a few of the many residues which make up an epitope are crucial for interaction and provide the critical contacts required for antibody recognition. This implies that antibody escape mutants are selected only if they contain changes at these crucial sites, or changes which introduce bulky side chains that sterically prevent antibody attachment. © 1993 Wiley-Liss, Inc.     

Appearance of neoantigen in mouse IgG1 upon reduction of interchain disulfide bridges: assessment of local and general conformational rearrangements using monoclonal antibody and small-angle X-ray scattering

Mouse hybridoma antibody E5D2 reacting with murine mono- and polyclonal IgG1 has been produced. MonAb E5D2 recognizes the antigenic determinant (epitope) buried in intact IgG1 and expressed upon mild reduction of interchain S-S bridges. Neither H nor L chains alone maintain epitope E5D2. Reassociation of gamma 1 chains (H chains of IgG1) with L chains results in complete restoration of this antigenic determinant. The data strongly suggest that epitope E5D2 depends on the quaternary structure of IgG1. The epitope is also expressed by reduced F(ab)2 fragment of IgG1 but is not connected with its antigen binding site. The likely localization of the epitope E5D2 is the interface between CH and CL domains. The second produced monAb F6C2 reacts with CH1-CL region of reduced mouse IgG2. Small-angle X-ray scattering experiments have demonstrated pronounced decrease of the radius of gyration of reduced IgG1 as compared to the intact one. This indicates general conformational changes of IgG1 molecule following mild reduction of Fab region S-S groups. Epitope E5D2 is the first quaternary antigenic subclass specific determinant described for C the region of mouse IgG. Thus, serologic expression of epitope E5D2 reveals precise conformational perturbations of small area near reduced S-S bridges while small-angle scattering demonstrates accompanying general transformation of IgG structure.     

2.9 A resolution structure of an anti-dinitrophenyl-spin-label monoclonal antibody Fab fragment with bound hapten

The crystal structure of the Fab fragment of the murine monoclonal anti-dinitrophenyl-spin-label antibody AN02 complexed with its hapten has been solved at 2.9 A resolution using a novel molecular replacement method. Prior to translation searches, a large number of the most likely rotation function solutions were subjected to a rigid body refinement against the linear correlation coefficient between intensities of observed and calculated structure factors. First, the overall orientation of the search model and then the orientations and positions of the four Fab domains (VH, VL, CH1 and CL) were refined. This procedure clearly identified the correct orientation of the search model. The refined search model was then subjected to translation searches which unambiguously determined the enantiomer and position in the unit cell of the crystal. The successful search model was refined 2.5 A crystal structure of the Fab fragment of HyHel-5 from which non-matching residues in the variable domains had been removed. HyHel-5 is a murine monoclonal antibody whose heavy and light chains are of the same subclass (gamma 1, kappa, respectively) as AN02. After molecular replacement the structure of the AN02 Fab has been refined using simulated annealing in combination with model building and conjugate gradient refinement to a current crystallographic R-factor of 19.5% for 12,129 unique reflections between 8.0 and 2.9 A. The root-mean-square (r.m.s.) deviation from ideal bond lengths is 0.014 A, and the r.m.s. deviation from ideal bond angles is 3.1 degrees. The electron density reveals the hapten sitting in a pocket formed by the loops of the complementarity determining region. The dinitrophenyl ring of the hapten is sandwiched between the indole rings of Trp96 of the heavy-chain and Trp91 of the light-chain. The positioning of the hapten and general features of the combining site are in good agreement with the results of earlier nuclear magnetic resonance experiments.     

Structure of Fab New at 6 Å Resolution

The structure of human immunoglobulin Fab′ fragment has been elucidated at a resolution of 6 Å. There are two globular subunits, which presumably correspond to the variable and constant regions of the heavy and light polypeptide chains of immunoglobulins     

Cloning, expression, and characterization of the Fab fragment of the anti-lysozyme antibody HyHEL-5

Hybridoma cDNAs encoding the individual chains of the Fab fragment of the well characterized murine monoclonal antibody HyHEL-5 were cloned and sequenced. The recombinant Fab fragment was produced by expressing each chain in a separate Escherichia coli pET vector, denaturing inclusion bodies and co-refolding. Characterization of the purified Fab by MALDI-TOF mass spectrometry and N-terminal amino acid sequencing demonstrated proper processing of the individual chains. The association of the recombinant Fab fragment with hen egg lysozyme and the avian epitope variant bobwhite quail lysozyme was found by isothermal titration calorimetry to have energetics very similar to that of the HyHEL-5 IgG. Heterologous expression of the HyHEL-5 Fab fragment opens the way to structure/function studies in this well-known system.     

Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains.

The display of proteins on the surface of phage offers a powerful means of selecting for rare genes encoding proteins with binding activities. Recently we found that antibody heavy and light chain variable (V) domains fused as a single polypeptide chain to a minor coat protein of filamentous phage fd, could be enriched by successive rounds of phage growth and panning with antigen. This allows the selection of antigen-binding domains directly from diverse libraries of V-genes. Now we show that heterodimeric Fab fragments can be assembled on the surface of the phage by linking one chain to the phage coat protein, and secreting the other into the bacterial periplasm. Furthermore by introducing an amber mutation between the antibody chain and the coat protein, we can either display the antibody on phage using supE strains of bacteria, or produce soluble Fab fragment using non-suppressor strains. The use of Fab fragments may offer advantages over single chain Fv fragments for construction of combinatorial libraries.