Asunto(s)
Bacteriófagos/genética , Proteínas de la Cápside , Cápside/genética , Biblioteca de Péptidos , Secuencia de Aminoácidos , Bacteriófagos/fisiología , Cápside/química , Cromatografía de Afinidad , Clonación Molecular/métodos , Vectores Genéticos , Genoma Viral , Intrones , Ligandos , Datos de Secuencia Molecular , Plásmidos , Pliegue de Proteína , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Mapeo Restrictivo , Virología/métodosRESUMEN
By using the selectively infective phage (SIP) technology, we selected non-repetitive linkers for a single-chain Fv fragment to have genes more robust against deletions in PCR-based gene assembly and directed evolution experiments than is the case for the classical (Gly4Ser)3 linker. We designed linkers encoding turns at both ends and random positions in the middle where glycines and polar and charged residues were allowed to occur. After only a single round of SIP, all clones obtained were fully functional. Properties such as antigen binding constants, urea denaturation curves and expression of soluble scFv fragments were identical with those of the parental fragment with the (Gly4Ser)3 linker. This demonstrates that SIP is a very fast and powerful technique to remove rapidly sequences of poor functionality, exclusively yielding sequences of the desired overall property in a single round.
Asunto(s)
Bacteriófagos/genética , Fragmentos de Inmunoglobulinas/biosíntesis , Región Variable de Inmunoglobulina/biosíntesis , Biblioteca de Péptidos , Conformación Proteica , Ingeniería de Proteínas , Secuencia de Aminoácidos , Afinidad de Anticuerpos , Bacteriófagos/inmunología , Secuencia de Bases , Sitios de Unión de Anticuerpos , Ensayo de Inmunoadsorción Enzimática , Fluoresceína-5-Isotiocianato , Glicina , Fragmentos de Inmunoglobulinas/química , Fragmentos de Inmunoglobulinas/genética , Región Variable de Inmunoglobulina/química , Región Variable de Inmunoglobulina/genética , Datos de Secuencia Molecular , Mutagénesis , Reacción en Cadena de la Polimerasa/métodos , Desnaturalización Proteica , Pliegue de Proteína , Proteínas Recombinantes de Fusión/biosíntesis , Serina , Solubilidad , UreaRESUMEN
We investigated which molecules are selected from a model library by the selectively infective phage (SIP) methodology. As a model system, we used the fluorescein binding single-chain Fv fragment FITC-E2, and from a 3D-model, we identified 11 residues potentially involved in hapten binding and mutated them individually to alanines. The binding constant of each mutant was determined by fluorescence titration, and each mutant was tested individually as well as in competitive SIP experiments for infectivity. After three rounds of SIP, only molecules with KD values within a factor of 2 of the tightest binder remain, and among those, a mutant no longer carrying an unnecessary exposed tryptophan residue is preferentially selected. SIP is shown to select for the best overall properties of the displayed molecules, including folding behavior, stability and affinity.
Asunto(s)
Anticuerpos/inmunología , Afinidad de Anticuerpos , Fragmentos de Inmunoglobulinas/química , Fragmentos de Inmunoglobulinas/inmunología , Pliegue de Proteína , Anticuerpos/química , Reacciones Antígeno-Anticuerpo , Colifagos/genética , Colifagos/fisiología , Escherichia coli/genética , Escherichia coli/virología , Fluoresceína-5-Isotiocianato , Haptenos/química , Haptenos/inmunología , Fragmentos de Inmunoglobulinas/genética , Región Variable de Inmunoglobulina/química , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/inmunología , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Biblioteca de Péptidos , Plásmidos , Espectrometría de FluorescenciaRESUMEN
We review here advances in the selectively infective phage (SIP) technology, a novel method for the in vivo selection of interacting protein-ligand pairs. A 'selectively infective phage' consists of two components, a filamentous phage particle made non-infective by replacing its N-terminal domains of gene3 protein (g3p) with a ligand-binding protein, and an 'adapter' molecule in which the ligand is linked to those N-terminal domains of g3p which are missing from the phage particle. Infectivity is restored when the displayed protein binds the ligand and thereby attaches the missing N-terminal domains of g3p to the phage particle. Phage propagation becomes strictly dependent on the protein-ligand interaction. This method shows promise both in the area of library screening and in the optimization of peptides or proteins.
Asunto(s)
Bacteriófagos/genética , Bacteriófagos/patogenicidad , Ingeniería de Proteínas , Bacteriófagos/química , Evolución Molecular , Ingeniería Genética , Humanos , Inoviridae/genética , Biblioteca de PéptidosRESUMEN
Selectively-infective phage (SIP) is a novel methodology for the in vivo selection of interacting protein-ligand pairs. It consists of two components, (1) a phage particle made non-infective by replacing its N-terminal domains of geneIII protein (gIIIp) with a ligand-binding protein, and (2) an "adapter" molecule in which the ligand is linked to those N-terminal domains of gIIIp which are missing from the phage particle. Infectivity is restored when the displayed protein binds to the ligand and thereby attaches the missing N-terminal domains of gIIIp to the phage particle. Phage propagation is thus strictly dependent on the protein-ligand interaction. We have shown that the insertion of beta-lactamase into different positions of gIIIp, mimicking the insertion of a protein-ligand pair, led to highly infective phage particles. Any phages lacking the first N-terminal domain were not infective at all. In contrast, those lacking only the second N-terminal domain showed low infectivity irrespective of the presence or absence of the F-pilus on the recipient cell, which could be enhanced by addition of calcium. An anti-fluorescein scFv antibody and its antigen fluorescein were examined as a protein-ligand model system for SIP experiments. Adapter molecules, synthesized by chemical coupling of fluorescein to the purified N-terminal domains, were mixed with non-infective anti-fluorescein scFv-displaying phages. Infection events were strictly dependent on fluorescein being coupled to the N-terminal domains and showed a strong dependence on the adapter concentration. Up to 10(6) antigen-specific events could be obtained from 10(10) input phages, compared to only one antigen-independent event. Since no separation of binders and non-binders is necessary, SIP is promising as a rapid procedure to select for high affinity interactions.
Asunto(s)
Inovirus , Ligandos , Biblioteca de Péptidos , Proteínas/metabolismo , Cloruro de Calcio/farmacología , Proteínas de la Cápside , Proteínas de Unión al ADN/genética , Escherichia coli/virología , Fluoresceína , Fluoresceínas , Vectores Genéticos/genética , Fragmentos de Inmunoglobulinas/genética , Fragmentos de Inmunoglobulinas/metabolismo , Inovirus/genética , Inovirus/patogenicidad , Cloruro de Magnesio/farmacología , Unión Proteica , Proteínas/genética , Proteínas Virales de Fusión/genética , beta-Lactamasas/genéticaRESUMEN
By constructing Fv and single-chain Fv (scFv) fragments of antibodies, the variable domains are taken out of their natural context in the Fab fragment, where they are associated with the constant domains of the light (CL) and heavy chain (CH1). As a consequence, all residues of the former variable/constant domain interface become solvent exposed. In an analysis of 30 non-redundant Fab structures it was found that at the former variable/constant domain interface of the Fv fragment the frequency of exposed hydrophobic residues is much higher than in the rest of the Fv fragment surface. We investigated the importance of these residues for different properties such as folding in vivo and in vitro, thermodynamic stability, solubility of the native protein and antigen affinity. The experimental model system was the scFv fragment of the anti-fluorescein antibody 4-4-20, of which only 2% is native when expressed in the periplasm of Escherichia coli. To improve its in vivo folding, a mutagenesis study of three newly exposed interfacial residues in various combinations was carried out. The replacement of one of the residues (V84D in VH) led to a 25-fold increase of the functional periplasmic expression yield of the scFv fragment of the antibody 4-4-20. With the purified scFv fragment it was shown that the thermodynamic stability and the antigen binding constant are not influenced by these mutations, but the rate of the thermally induced aggregation reaction is decreased. Only a minor effect on the solubility of the native protein was observed, demonstrating that the mutations prevent aggregation during folding and not of the native protein. Since the construction of all scFv fragments leads to the exposure of these residues at the former variable/constant domain interface, this strategy should be generally applicable for improving the in vivo folding of scFv fragments and, by analogy, also the in vivo folding of other engineered protein domains.
Asunto(s)
Fragmentos de Inmunoglobulinas/química , Región Variable de Inmunoglobulina/química , Pliegue de Proteína , Secuencia de Aminoácidos , Mapeo Epitopo , Enlace de Hidrógeno , Fragmentos de Inmunoglobulinas/genética , Región Variable de Inmunoglobulina/genética , Modelos Moleculares , Datos de Secuencia Molecular , Mutación Puntual , Proteínas Recombinantes/química , TermodinámicaRESUMEN
Using a cell-bound immunogen, we have generated a monoclonal antibody, 3D5, that recognizes carboxy-terminal oligo-histidine tags (His tags) on a wide variety of proteins. From this monoclonal antibody, we have generated a single-chain fragment of the variable domains (scFv), a dimeric scFv-alkaline phosphatase fusion and an oligovalent scFv-display phage. The antibody in its various formats is an effective tool used in fluorescence-activated cell sorting analysis, the BIAcore method, Western blots and enzyme-linked immunosorbent assay (ELISA). Western blots and ELISAs can be developed directly by using crude extracts of E.coli cells that produce the scFv-alkaline phosphatase fusion, thus providing an inexhaustable and convenient supply of detection reagent. Alternatively, oligovalent scFv-displaying phage can be used directly from culture supernatants for this purpose. The dissociation constants, KD of the peptide KGGHHHHH (KD = 4 x 10(-7) M) and of imidazole (KD = 4 x 10(-4) M) were determined. Molecular modeling of the Fv fragment suggests the occurrence of two salt bridges between the protonated histidine side chains of the peptide and the acidic groups in the antibody, explaining why the antibody or the substrate may be eluted under mildly basic conditions.
Asunto(s)
Anticuerpos Monoclonales , Bacteriófagos/genética , Histidina/metabolismo , Proteínas Recombinantes/química , Western Blotting , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/química , Escherichia coli/metabolismo , Citometría de Flujo/métodos , Histidina/inmunología , Fragmentos de Inmunoglobulinas , Indicadores y Reactivos , Unión ProteicaRESUMEN
Several antibody-dependent mechanisms have been postulated to mediate neutralization of different animal viruses, including blocking of docking to receptors, induction of conformational changes in the virus coat, and Fc-dependent opsonization. We have studied the molecular requirements for antibody-mediated neutralization of vesicular stomatitis virus (VSV) in vitro and protection against lethal disease in vivo with a single-chain Fv fragment (scFv) and the corresponding bivalent miniantibody (scFv-dHLX) generated from a VSV-neutralizing monoclonal antibody. Both monovalent scFv and bivalent scFv-dHLX miniantibodies were able to neutralize VSV in vitro and to protect interferon-alphabeta receptor-deficient (IFN-alphabeta R-/-) mice against lethal disease after intravenous injection of 50 plaque-forming units (pfu) VSV pre-incubated with the scFv reagents. Similarly, severe-combined immunodeficient (SCID) mice infected with immune complexes of 10(8) pfu VSV and bivalent scFv-dHLX were protected against lethal disease; however, mice infected with immune complexes of 10(8) pfu VSV and monovalent scFv were not. Although repeated scFv-dHLX treatment reduced virus quantities in the blood, neither SCID nor IFN-alphabeta R-/- mice were protected against lethal disease after passive immunization and subsequent VSV infection. This was due to the short half-life of 17 min of scFv-dHLX in the circulation. These data demonstrate that neutralization of VSV and protection against lethal disease do not require Fc-mediated mechanisms and that cross-linking is not crucial for protection against physiologically relevant virus doses in vivo.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Fragmentos de Inmunoglobulinas/inmunología , Región Variable de Inmunoglobulina/inmunología , Infecciones por Rhabdoviridae/mortalidad , Infecciones por Rhabdoviridae/prevención & control , Virus de la Estomatitis Vesicular Indiana/inmunología , Secuencia de Aminoácidos , Animales , Unión Competitiva/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Datos de Secuencia MolecularRESUMEN
We have developed a chloramphenicol resistant derivative of fd phage with which cognate pairs of antibodies and antigens can be selected. The phage genome encodes a fusion of single-chain antibody to the C-terminal domain of gIIIp, rendering the phage non-infective. The antigen fused to the N-terminal domains of gIIIp is encoded in the same phage genome. Antigen and antibody fusion interact with each other in the periplasm of the phage-producing cell, restoring infectivity. This system has a very low background and will allow simultaneous randomisation of antibody and antigen.
Asunto(s)
Complejo Antígeno-Anticuerpo/inmunología , Proteínas de Unión al ADN/inmunología , Inovirus/genética , Proteínas Virales de Fusión , Proteínas Virales/inmunología , Secuencia de Aminoácidos , Complejo Antígeno-Anticuerpo/genética , Proteínas de la Cápside , Cloranfenicol , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Vectores Genéticos , Genoma Viral , Fragmentos de Inmunoglobulinas/genética , Fragmentos de Inmunoglobulinas/inmunología , Inovirus/inmunología , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
The Fv and Fab fragment and both orientations of the single-chain Fv fragment (VH-linker-VL and VL-linker-VH) of an antibody can be expressed in functional form in the periplasm of Escherichia coli, but the yield of these correctly assembled proteins is limited by the periplasmic folding process. While the periplasmic E. coli disulfide isomerase DsbA is required for this assembly, its functional over-expression does not significantly change the folding limit. Similarly, the functionally over-expressed E. coli proline cis-trans isomerase does not change the amount of all but one of the antibody fragments, not even if DsbA is over-expressed as well. Therefore, aggregation steps in the periplasm appear to compete with periplasmic folding, and they may occur before disulfide formation and/or proline cis-trans isomerization takes place and be independent of their extent.