An S-layer heavy chain camel antibody fusion protein for generation of a nanopatterned sensing layer to detect the prostate-specific antigen by surface plasmon resonance technology

The bacterial cell surface layer (S-layer) protein of Bacillus sphaericus CCM 2177 assembles into a square lattice structure and recognizes a distinct type of secondary cell wall polymer (SCWP) as the proper anchoring structure in the rigid cell wall layer. For generating a nanopatterned sensing layer with high density and well defined distance of the ligand on the outermost surface, an S-layer fusion protein incorporating the sequence of a variable domain of a heavy chain camel antibody directed against prostate-specific antigen (PSA) was constructed, produced, and recrystallized on gold chips precoated with thiolated SCWP. The S-layer protein moiety consisted of the N-terminal part which specifically recognized the SCWP as binding site and the self-assembly domain. The PSA-specific variable domain of the camel heavy chain antibody was selected by several rounds of panning from a phage display library of an immunized dromedary, and was produced by heterologous expression in Escherichia coli. For construction of the S-layer fusion protein, the 3'-end of the sequence encoding the C-terminally truncated form rSbpA(31)(-)(1068) was fused via a short linker to the 5'-end of the sequence encoding cAb-PSA-N7. The S-layer fusion protein had retained the ability to self-assemble into the square lattice structure. According to the selected fusion site in the SbpA sequence, the cAb-PSA-N7 moiety remained located on the outer surface of the protein lattice. After recrystallization of the S-layer fusion protein on gold chips precoated with thiolated SCWP, the monomolecular protein lattice was exploited as sensing layer in surface plasmon resonance biochips to detect PSA.     

Construction of a functional S-layer fusion protein comprising an immunoglobulin G-binding domain for development of specific adsorbents for extracorporeal blood purification

The chimeric gene encoding a C-terminally-truncated form of the S-layer protein SbpA from Bacillus sphaericus CCM 2177 and two copies of the Fc-binding Z-domain was constructed, cloned, and heterologously expressed in Escherichia coli HMS174(DE3). The Z-domain is a synthetic analogue of the B-domain of protein A, capable of binding the Fc part of immunoglobulin G (IgG). The S-layer fusion protein rSbpA(31-1068)/ZZ retained the specific properties of the S-layer protein moiety to self-assemble in suspension and to recrystallize on supports precoated with secondary cell wall polymer (SCWP), which is the natural anchoring molecule for the S-layer protein in the bacterial cell wall. Due to the construction principle of the S-layer fusion protein, the ZZ-domains remained exposed on the outermost surface of the protein lattice. The binding capacity of the native or cross-linked monolayer for human IgG was determined by surface plasmon resonance measurements. For batch adsorption experiments, 3-microm-diameter, biocompatible cellulose-based, SCWP-coated microbeads were used for recrystallization of the S-layer fusion protein. In the case of the native monolayer, the binding capacity for human IgG was 5.1 ng/mm(2), whereas after cross-linking with dimethyl pimelimidate, 4.4 ng of IgG/mm(2) was bound. This corresponded to 78 and 65% of the theoretical saturation capacity of a planar surface for IgGs aligned in the upright position, respectively. Compared to commercial particles used as immunoadsorbents to remove autoantibodies from sera of patients suffering from an autoimmune disease, the IgG binding capacity of the S-layer fusion protein-coated microbeads was at least 20 times higher. For that reason, this novel type of microbeads should find application in the microsphere-based detoxification system.     

Isolation and characterization of an active variable domain from a homogeneous rabbit antibody light chain.

The variable domain (VL) of allotype b4 light chains of rabbit IgG was isolated from both nonimmune heterogeneous IgG and a homogeneous antibody directed against type III pneumococcal polysaccharide. Light chains were first isolated and then cleaved under mild acidic conditions between residues 109 and 110. Reduction with dithiothreitol in guanidine hydrochloride cleaved both intradomain disulfide bridges as well as the interdomain disulfide bridge joining the variable and constant domain. The sulfhydryl groups were protected after reduction by p-chloromercuribenzoate. VL was isolated from this mixture of variable and constant domains by affinity chromatography, utilizing sheep antibodies directed against a peptide including residues 110--211 from nonimmune IgG light chain. The isolated VL domain was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and automated Edman degradation. VL from a homogeneous antibody was treated with dithiothreitol to remove p-chloromercuribenzoate, reoxidized, and recombined with homologous heavy chain. The binding of this recombinant to type III pneumococcal polysaccharide was identical with that of the light-chain--heavy-chain recombinant.     

15N, 13C and 1H resonance assignments and secondary structure determination of a variable heavy domain of a heavy chain antibody

Heavy chain antibodies differ in structure to conventional antibodies lacking both the light chain and the first heavy chain constant domain (CH1). Characteristics of the antigen-binding variable heavy domain of the heavy chain antibody (VHH) including the smaller size, high solubility and stability make them an attractive alternative to more traditional antibody fragments for detailed NMR-based structural analysis. Here we report essentially complete backbone and side chain ¹⁵N, ¹³C and ¹H assignments for a free VHH. Analysis of the backbone chemical shift data obtained indicates that the VHH is comprised predominantly of β-sheets corresponding to nearly 60 % of the protein backbone.     

Construction of a large phage-displayed human antibody domain library with a scaffold based on a newly identified highly soluble, stable heavy chain variable domain

Currently, almost all U.S. Food and Drug Administration-approved therapeutic antibodies and the vast majority of those in clinical trials are full-size antibodies mostly in an immunoglobulin G1 format of about 150 kDa in size. Two fundamental problems for such large molecules are their poor penetration into tissues (e.g., solid tumors) and poor or absent binding to regions on the surface of some molecules [e.g., on the human immunodeficiency virus envelope glycoprotein (Env)] that are accessible by molecules of smaller size. We have identified a phage-displayed heavy chain-only antibody by panning of a large (size, ∼ 1.5 × 10¹⁰) human naive Fab (antigen-binding fragment) library against an Env and found that the heavy chain variable domain (V_H) of this antibody, designated as m0, was independently folded, stable, highly soluble, monomeric, and expressed at high levels in bacteria. m0 was used as a scaffold to construct a large (size, ∼ 2.5 × 10¹⁰), highly diversified phage-displayed human V_H library by grafting naturally occurring complementarity-determining regions (CDRs) 2 and 3 of heavy chains from five human antibody Fab libraries and by randomly mutating four putative solvent-accessible residues in CDR1 to A, D, S, or Y. The sequence diversity of all CDRs was determined from 143 randomly selected clones. Most of these V_Hs were with different CDR2 origins (six of seven groups of V_H germlines) or CDR3 lengths (ranging from 7 to 24 residues) and could be purified directly from the soluble fraction of the Escherichia coli periplasm. The quality of the library was also validated by successful selection of high-affinity V_Hs against viral and cancer-related antigens; all selected V_Hs were monomeric, easily expressed, and purified with high solubility and yield. This library could be a valuable source of antibodies targeting size-restricted epitopes and antigens in obstructed locations where efficient penetration could be critical for successful treatment.     

Phage antibody fragments library combining a single human light chain variable region with immune mouse heavy chain variable regions

We describe the construction of a phage antibody fragments library which combines, in a single cloning step, a synthetic human light chain variable region (V(L)) with a diverse set of heavy chain variable regions, from a mouse immunized with the prostate specific antigen (PSA). Despite V(L) restriction, selection from this library rendered two different single chain Fv antibody fragments, specifically recognizing PSA. The human V(L), used as a general partner for mouse heavy chains, was constructed by linking the germline A27 gene and the J(K)1 minigene segment, both of which are prominently involved in human antibody responses. Our approach offers a fast and simple way to produce half-human molecules, while keeping the advantage of immunizing animals for high affinity antibodies.     

A simple method for midkine purification by affinity chromatography with a heavy chain variable domain (VH) fragment of antibody

A DNA fragment for a heavy chain variable domain (VH) was prepared from a hybridoma that produces a monoclonal antibody against human midkine (MK). The antibody fragment was produced in Escherichia coli and its affinity for chemically synthesized full length MK or recombinant midkine c-terminus (MKc-half) protein was confirmed by ELISA. An Escherichia coli cell lysate expressing MKc-half was applied to a VH fragment-coupled Sepharose 4B column and eluted with a buffer containing 0.5 M NaCl. SDS-PAGE analysis revealed a high degree of purity of the MKc-half protein in the eluent, showing the utility of a recombinant VH fragment in purification of proteins by affinity chromatography.     

Development and characterization of a novel fusion protein composed of a human IgG1 heavy chain constant region and a single-chain fragment variable antibody against Venezuelan equine encephalitis virus

Murine monoclonal antibody 1A4A1 has been shown to recognize a conserved neutralizing epitope of envelope glycoprotein E2 of Venezuelan equine encephalitis virus. It is a potential candidate for development of a second generation antibody for both immunodiagnosis and immunotherapy. In order to minimize the immunogenicity of murine antibodies and to confer human immune effector functions on murine antibodies, a recombinant gene fusion was constructed. It encoded a human IgG1 heavy chain constant region and a single-chain fragment variable antibody of 1A4A1. After expression in bacteria as inclusion bodies, the recombinant antibody was purified and refolded in vitro. The recombinant soluble antibody was demonstrated to retain high antigen-binding affinity to Venezuelan equine encephalitis virus and to possess some human IgG crystallizable fragment domain functions, such as recognition by protein G and human complement C1q binding. On non-reducing and reducing gel electrophoresis analysis of proteolytic fragments of the recombinant antibody, disulfide bond formation was found in the hinge region of the antibody. From these data, it was concluded that the recombinant antibody was capable of antigen recognition, and retained several functional activities. This work forms the basis for characterization of the recombinant antibody as to efficacy in vivo.     

Chemical basis for the affinity maturation of a camel single domain antibody

Affinity maturation of classic antibodies supposedly proceeds through the pre-organization of the reactive germ line conformational isomer. It is less evident to foresee how this can be accomplished by camelid heavy-chain antibodies lacking light chains. Although these antibodies are subjected to somatic hypermutation, their antigen-binding fragment consists of a single domain with restricted flexibility in favor of binding energy. An antigen-binding domain derived from a dromedary heavy-chain antibody, cAb-Lys3, accumulated five amino acid substitutions in CDR1 and CDR2 upon maturation against lysozyme. Three of these residues have hydrophobic side chains, replacing serines, and participate in the hydrophobic core of the CDR1 in the mature antibody, suggesting that conformational rearrangements might occur in this loop during maturation. However, transition state analysis of the binding kinetics of mature cAb-Lys3 and germ line variants show that the maturation of this antibody relies on events late in the reaction pathway. This is reflected by a limited perturbation of k(a) and a significantly decreased k(d) upon maturation. In addition, binding reactions and the maturation event are predominantly enthalpically driven. Therefore, maturation proceeds through the increase of favorable binding interactions, or by the reduction of the enthalpic penalty for desolvation, as opposed to large entropic penalties associated with conformational changes and structural plasticity. Furthermore, the crystal structure of the mutant with a restored germ line CDR2 sequence illustrates that the matured hydrophobic core of CDR1 in cAb-Lys3 might be compensated in the germ line precursor by burying solvent molecules engaged in a stable hydrogen-bonding network with CDR1 and CDR2.     

人源抗EPO基因工程抗体重链可变区的克隆和鉴定

利用噬菌体抗体显示技术筛选 EPO的人源抗体 ,得到了抗 EPO的人源抗体的重链基因。此抗体基因在噬菌体表面呈现的抗体分子具有良好的抗体活性和特异性。为制备完整的、具有更高亲和力的抗体打下了基础。     

Solution structure and backbone dynamics of an antigen-free heavy chain variable domain (VHH) from Llama

Camelids, (dromedaries, camels, and llamas) produce heavy-chains antibodies, with their antigen recognition sites composed of a single VH-like domain, referred to as VHH. The solution structure of one of these VHHs domains (VHH-H14), raised against the alpha subunit of the human chorionic gonadotropin hormone (hCG), has been determined by (15)N heteronuclear three-dimensional NMR spectroscopy. The framework is well resolved within the set of 20 best-calculated NMR structures and is close to that of classical VH domains from vertebrate antibodies, consisting of two antiparallel beta-sheets organized in a beta-barrel. Loops display a lower precision, especially the Complementarity Determining Regions (CDRs), involved in antigen recognition. Comparison of the three-dimensional VHH-H14 solution structure with its previously solved crystal structure (Spinelli et al., Nature Struct. Biol. 1996;3:752-757) reveals a high similarity to the framework, whereas significant conformational differences occur on CDRs, leading to the assumption that the antigen recognition site is a more mobile part. In order to deepen our insights into the dynamics of VHH-H14 in solution, (15)N relaxation was measured with longitudinal R1 and transverse R2 self-relaxation rates, and (15)N steady-state heteronuclear nuclear Overhauser enhancements (NOE), making it possible to probe picosecond-to-millisecond internal motions. Determination of dynamic parameters (S(2), tau(e), and Rex) through the Lipari-Szabo Model-free approach enables the identification of several regions with enhanced dynamics. Especially, the mobility measurements from NMR confirm that the antigen recognition site is the most mobile part of the VHH-H14 domain on picosecond-to-nanosecond fast time scales. Several residues belonging to the three CDRs are submitted to chemical exchange processes occurring on slow microsecond-to-millisecond time scales, suggesting that the formation of the VHH/antigen complex should be accompanied by structural changes.     

Limited diversity of the immunoglobulin heavy chain variable domain of the emerald rockcod Trematomus bernacchii

To investigate the diversity of the immunoglobulin heavy chain variable domain of the cold adapted teleost Trematomus bernacchii, 45 cDNA clones, containing complete or partial sequences of rearranged VH/D/JH segments, were analysed. Clones were isolated from a spleen library constructed by 5' RACE or from an expression library previously constructed and immunoscreened with rabbit anti- T. bernacchii Ig heavy chain antibodies. VH sequences shared, on average, 79.9% nucleotide identity and defined only two gene families referred to as Trbe VH I and Trbe VH II, the latter comprising 89% of the VH sequences analysed in this study. A Southern blot analysis, performed with family specific probes, revealed that there are at least 25 genomic VH genes. A phylogenetic tree showed that Trbe VH I clustered with VH genes belonging to group D and Trbe VH II with those of group C. Four putative distinct D segments were found to contribute to the diversity of CDR3, which showed a high glycine content. The Shannon analysis revealed that FRs are very highly conserved. Of CDRs, CDR2 exhibits a mean entropy value higher than CDR1, contributing to variability in a significant manner. Moreover, eight distinct JH segments were identified. These findings provide several clues suggesting a limited diversity of the VH genes in the Antarctic teleost T. bernacchii.     

Production of a functional catalytic antibody ScFv-NusA fusion protein in bacterial cytoplasm

Functional expression of catalytic antibodies in the cytoplasm of E. coli is potentially of great interest in searching for new catalysts by genetic selection. Herein, a catalytic antibody single chain Fv (ScFv) 14D9, which catalyzes a highly enantioselective protonation, was expressed as a NusA fusion protein under the T7 promoter. A functional disulfide-containing ScFv fusion protein was obtained in the oxidizing environment of bacterial cytoplasm. The 14D9 ScFv could not be overexpressed alone without NusA fusion. The highly soluble NusA protein most likely retards aggregate formation of ScFv and indirectly supports correct folding and disulfide bridge formation in the fusion construct ScFv-NusA. The ScFv-NusA fusion product shows highly enantioselective, specific, hapten inhibited catalytic activity comparable to its parent monoclonal antibody, 14D9. The NusA fusion method might be generally helpful for functional antibody expression in vivo and for the new development of biocatalysts by genetic selection.     

Site-specific integration of light chain and heavy chain genes of antibody into CHO-K1 stable hot spot and detection of antibody and fusion protein expression level

Abstract

Expression cell line constructed by random integration method will often meet with unstable expression problem because target genes may be integrated into unstable region of chromatin. Rational cell line construction can overcome this shortcoming by inserting target gene into stable region of chromatin specifically. Here, we successfully got one knock-in cell line where light chain and heavy chain genes of antibody was site specifically integrated into stable hot spot reported before via homologous dependent recombination method mediated by CRISPR/Cas9. The targeting efficiency was around 1.35%. This cell line together with other three pre-established targeting cell lines (targeting with glucagon-like peptide 1 with human serum albumin fusion protein gene, or NGGH) were all undergoing protein expression level detection. In adherent cell mode, the amount of antibody expressed per cell per day were all around 0.006?pg/cell/day over passage 3, 12, 23, 35 and 50 while the amount of NGGH expressed per cell per day of 3 cell lines were all around 1.2?pg/cell/day over passage 3, 12, 23, 35 and 50. In batch mode, the antibody concentration within supernatant were around 2.5?µg/L over passage 1, 25, and 50 while the NGGH fusion protein concentration within supernatant were around 17?mg/L over passage 1, 25, and 50.     

Identification of a membrane fusion domain and an oligomerization domain in the baculovirus GP64 envelope fusion protein.

The baculovirus GP64 envelope fusion protein (GP64 EFP) is the major envelope glycoprotein of the budded virion and has been shown to mediate acid-triggered membrane fusion both in virions and when expressed alone in transfected cells. Using site-directed mutagenesis and functional assays for oligomerization, transport, and membrane fusion, we localized two functional domains of GP64 EFP. To identify a fusion domain in the GP64 EFP of the Orgyia pseudotsugata multiple nuclear polyhedrosis virus (OpMNPV), we examined two hydrophobic regions in the GP64 EFP ectodomain. Hydrophobic region I (amino acids 223 to 228) is a cluster of 6 hydrophobic amino acids exhibiting the highest local hydrophobicity in the ectodomain. Hydrophobic region II (amino acids 330 to 338) lies within a conserved region of GP64 EFP that contains a heptad repeat of leucine residues and is predicted to form an amphipathic alpha-helix. In region I, nonconservative amino acid substitutions at Leu-226 and Leu-227 (at the center of the hydrophobic cluster) completely abolished fusion activity but did not prevent GP64 EFP oligomerization or surface localization. To confirm the role of region I in membrane fusion activity, we used a synthetic 21-amino-acid peptide to generate polyclonal antibodies against region I and demonstrated that antipeptide antibodies were capable of both neutralizing membrane fusion activity and reducing infectivity of the virus. In hydrophobic region II, mutations were designed to disrupt several structural characteristics: a heptad repeat of leucine, a predicted alpha-helix, or the local hydrophobicity along one face of the helix. Single alanine substitutions for heptad leucines did not prevent oligomerization, transport, or fusion activity. However, multiple alanine substitutions or proline (helix-destabilizing) substitutions disrupted both oligomerization and transport of GP64 EFP. In addition, a deletion that removed region II and the predicted alpha-helix was defective for oligomerization, whereas a larger deletion that retained region II and the predicted helix was oligomerized. These results indicate that region II is required for oligomerization and transport and suggest that the predicted helical structure of this region may be important for this function. Thus, by using mutagenesis, functional assays, and antibody inhibition, two functional domains were localized within the baculovirus GP64 EFP: a fusion domain located at amino acids 223 to 228 and an oligomerization domain located at amino acids 327 to 335 within a predicted amphipathic alpha-helix.     

An antibody single-domain phage display library of a native heavy chain variable region: isolation of functional single-domain VH molecules with a unique interface.

To develop very small antibody-derived recognition units for experimental, medical, and drug design purposes, a heavy chain variable region (VH) single-domain phage-display library was designed and constructed. The scaffold that was used for library construction was a native sequence of a monoclonal antibody with a unique VH/VL interface. There was no need to modify any residues in the VL interface to avoid non-specific binding of VH domain. The library repertoire, consisting of 4x10(8)independent clones, was generated by the randomization of nine amino acid residues in complementary determining region 3. The library was screened by binding to protein antigens, and individual clones were isolated. The VH genes encoding for specific binding clones were rescued and large amounts of soluble and stable single-domain VH protein were made from insoluble inclusion bodies by in vitro refolding and purification. Biochemical and biophysical characterization of the VH protein revealed a highly specific, correctly folded, and stable monomeric molecule. Binding studies demonstrated an affinity of 20 nM. The properties of these molecules make them attractive for clinical, industrial, and research applications, as well as a step toward improvement in the design of small molecules that are based on the hypervariable loops of antibodies.     

Engineering and characterization of a single chain surrogate light chain variable domain

The surrogate light chain (SLC) is a key regulator of B cell development in the bone marrow, resulting in mature B cells that produce antibodies that are capable of interacting with antigens. The SLC comprises two noncovalently interacting proteins: VpreB and 14.1. We engineered a construct to represent the complete immunoglobulin-like domain of the SLC variable domain in a single protein chain that could be bacterially expressed. In this construct, the incomplete immunoglobulin domain of VpreB (residues 1-102) was linked to the J-segment of 14.1 (residues 40-53), which provided one beta-strand to complete the V-like domain (VpreBJ). Because VpreBJ has the interface to VH chains, but lacks the unique region of 14.1, which is important for SLC signaling, we predict that a properly folded VpreBJ would have the potential to act as a dominant negative mutant of the surrogate light chain. X-ray crystallography of VpreBJ at 2.0 A resolution showed that the engineering was successful. With its two beta-pleated sheets, packed face-to-face, the single chain VpreBJ resembles a mature light chain immunoglobulin V-domain (VL). The surface that would normally interact with the VH chain interacts with a crystallographically related VpreBJ molecule. The presence of dimeric species in solution was verified by analytical ultracentrifugation. VpreBJ is easily overexpressed in bacteria, while retaining the native conformation of an immunoglobulin domain, and thus may serve as an important reagent for future studies in B-cell development.     

Structure of a shark IgNAR antibody variable domain and modeling of an early-developmental isotype

The new antigen receptor (IgNAR) antibodies from sharks are disulphide bonded dimers of two protein chains, each containing one variable and five constant domains. Three types of IgNAR variable domains have been discovered, with Type 3 appearing early in shark development and being overtaken by the antigen-driven affinity-matured Type 1 and 2 response. Here, we have determined the first structure of a naturally occurring Type 2 IgNAR variable domain, and identified the disulphide bond that links and stabilizes the CDR1 and CDR3 loops. This disulphide bridge locks the CDR3 loop in an "upright" conformation in contrast to other shark antibody structures, where a more lateral configuration is observed. Further, we sought to model the Type 3 isotype based on the crystallographic structure reported here. This modeling indicates (1) that internal Type 3-specific residues combine to pack into a compact immunoglobulin core that supports the CDR loop regions, and (2) that despite apparent low-sequence variability, there is sufficient plasticity in the CDR3 loop to form a conformationally diverse antigen-binding surface.     

Sublingual vaccination with fusion protein consisting of the functional domain of hemagglutinin A of Porphyromonas gingivalis and Escherichia coli maltose-binding protein elicits protective immunity in the oral cavity

This study demonstrated that sublingual immunization with a fusion protein, 25k-hagA-MBP, which consists of a 25-kDa antigenic region of hemagglutinin A purified from Porphyromonas gingivalis fused to maltose-binding protein (MBP) originating from Escherichia coli as an adjuvant, elicited protective immune responses. Immunization with 25k-hagA-MBP induced high levels of antigen-specific serum IgG and IgA, as well as salivary IgA. High level titers of serum IgG and IgA were also induced for almost 1 year. In an IgG subclass analysis, sublingual immunization with 25k-hagA-MBP induced both IgG1 and IgG2b antibody responses. Additionally, numerous antigen-specific IgA antibody-forming cells were detected from the salivary gland 7 days after the final immunization. Mononuclear cells isolated from submandibular lymph nodes (SMLs) showed significant levels of proliferation upon restimulation with 25k-hagA-MBP. An analysis of cytokine responses showed that antigen-specific mononuclear cells isolated from SMLs produced significantly high levels of IL-4, IFN-γ, and TGF-β. These results indicate that sublingual immunization with 25k-hagA-MBP induces efficient protective immunity against P. gingivalis infection in the oral cavity via Th1-type and Th2-type cytokine production.