RESUMEN
Affinity maturation is often applied to improve the properties of antibodies isolated from universal antibody libraries in vitro. A synthetic human scFv antibody library was constructed in single immunoglobulin framework to enable rapid affinity maturation by updated Kunkel's mutagenesis. The initial diversity was generated predominantly in the V(H) domain combined with only 36 V(L) domain variants yielding 3 × 10(10) unique members in the phage-displayed library. After three rounds of panning the enriched V(H) genes from the primary library selections against lysozyme were incorporated into a ready-made circular single-stranded affinity maturation library containing 7 × 10(8) V(L) gene variants. Several unique antibodies with 0.8-10 nM (K(d), dissociation constant) affinities against lysozyme were found after panning from the affinity maturation library, contrasted by only one anti-lysozyme scFv clone with K(d) <20 nM among the clones panned from the primary universal library. The presented single-framework strategy provides a way to convey significant amount of functional V(H) domain diversity to affinity maturation without bimolecular ligation leading to a diverse set of antibodies with binding affinities in the low nanomolar range.
Asunto(s)
Afinidad de Anticuerpos/genética , Barajamiento de ADN/métodos , Biblioteca de Péptidos , Ingeniería de Proteínas/métodos , Anticuerpos de Cadena Única/genética , Secuencia de Aminoácidos , Humanos , Datos de Secuencia Molecular , Muramidasa/genética , Muramidasa/inmunología , Muramidasa/metabolismo , Mutagénesis , Pliegue de Proteína , Reproducibilidad de los Resultados , Anticuerpos de Cadena Única/inmunología , Anticuerpos de Cadena Única/metabolismoRESUMEN
The crystal structure of a Fab fragment of an anti-17beta-estradiol antibody 57-2 was determined in the absence and presence of the steroid ligand, 17beta-estradiol (E2), at 2.5 and 2.15-A resolutions, respectively. The antibody binds the steroid in a deep hydrophobic pocket formed at the interface between the variable domains. No major structural rearrangements take place upon ligand binding; however, a large part of the heavy chain variable domain near the binding pocket is unusually flexible and is partly stabilized when the steroid is bound. The nonpolar steroid skeleton of E2 is recognized by a number of hydrophobic interactions, whereas the two hydroxyl groups of E2 are hydrogen-bonded to the protein. Especially, the 17-hydroxyl group of E2 is recognized by an intricate hydrogen bonding network in which the 17-hydroxyl itself forms a rare four-center hydrogen bond with three polar amino acids; this hydrogen bonding arrangement accounts for the low cross-reactivity of the antibody with other estrogens such as estrone. The CDRH3 loop plays a prominent role in ligand binding. All the complementarity-determining regions of the light chain make direct contacts with the steroid, even CDRL2, which is rarely directly involved in the binding of haptens.
Asunto(s)
Estradiol/química , Fragmentos Fab de Inmunoglobulinas/química , Proteínas Recombinantes/química , Secuencia de Aminoácidos , Aminoácidos/química , Cristalografía por Rayos X , Humanos , Hidrógeno/metabolismo , Enlace de Hidrógeno , Ligandos , Modelos Químicos , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , Estructura Terciaria de ProteínaRESUMEN
The recombinant Fab fragment of the anti-17beta-oestradiol antibody 57-2 has been a target for several protein-engineering experiments. A method for production, purification and crystallization of the Fab fragment alone (apo form) and in complex with the major female sex hormone 17beta-oestradiol is reported here. Diffracting apo-form crystals were only obtained with microseeding; crystals of the Fab-steroid complex were produced by co-crystallization in the presence of oestradiol and cross-seeding with the apo-form crystals. The crystals were grown using vapour-diffusion methods with reservoir solutions containing 10-14% PEG 4000 or 8-12% PEG 8000 and Tris-HCl buffer at high pH (9.0-9.5). Both the apo and complex crystals belong to space group P2(1)2(1)2(1) and diffract to 2.0 A resolution. High-resolution X-ray data sets suitable for structure determination were collected from flash-cooled crystals using 25% glycerol as the cryoprotectant.
Asunto(s)
Estradiol/química , Fragmentos Fab de Inmunoglobulinas/química , Cristalografía por Rayos X , Escherichia coli , Fragmentos Fab de Inmunoglobulinas/genética , Fragmentos Fab de Inmunoglobulinas/aislamiento & purificación , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
Recombinant antibodies often contain N-terminal mutations arising from the use of degenerate cloning primer sets and/or the introduction of restriction sites in the framework 1 regions. We studied the effects of such mutations in a recombinant anti-estradiol Fab fragment derived from the hybridoma cell line 57-2. The 5' ends of the heavy and light chain genes were originally modified to introduce the restriction sites XhoI and SacI, respectively, for cloning purposes. However, the affinity and specificity of the recombinant Fab were lowered compared to the proteolytic Fab' fragment of the parental hybridoma IgG. Replacing the mutated sites with authentic amino acid coding sequences restored the binding properties as well as increased the bacterial production levels fivefold and 10-fold at 30 and 37 degrees C, respectively. Local changes in the antigen binding site were probed by determining the affinity constants (Kd) for estradiol and four related steroids. It was found that the mutated heavy chain amino terminus specifically increased the Kd for testosterone whereas the mutated light chain amino terminus decreased the Kd for all of the steroids to the same extent; the heavy and light chain effects were additive. Analysis of a newly determined crystal structure of the authentic Fab 57-2 in complex with estradiol suggests that mutations in the residue 2 in V(H), and 2 and 4 in the V(L) domain were those responsible for the observed effects. Their general roles as structure-determining residues for the CDR3 loops imply that similar effects can occur with other recombinant antibodies as well.
Asunto(s)
Estradiol/inmunología , Haptenos/metabolismo , Fragmentos Fab de Inmunoglobulinas/metabolismo , Mutación , Sustitución de Aminoácidos , Afinidad de Anticuerpos/genética , Especificidad de Anticuerpos/genética , Sitios de Unión/genética , Regiones Determinantes de Complementariedad/metabolismo , Estradiol/metabolismo , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/genética , Modelos Moleculares , Unión Proteica/genética , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Esteroides/inmunología , Esteroides/metabolismoRESUMEN
The length of the heavy chain complementarity-determining region 2 (CDRH2) was extended beyond what is found in germline genes to improve the binding properties of an anti-estradiol antibody. The previous immunochemical characterization and the molecular modeling of the high affinity (Ka=3.9x10(8)) murine anti-estradiol antibody 57-2 suggested that a part of the antigen was loosely recognized by the antibody. The CDRH2, because of its close location but scarce contacts with the hapten, was considered as a conceivable target for mutagenesis. Libraries with either two, three or four random amino acid insertions in the tip of the CDRH2 loop were constructed and displayed on the M13 filamentous phage as Fab fragments. Mutations were introduced also into the rest of the VHdomain by error-prone polymerase chain reaction to allow the surrounding structures to adapt to the extended CDRH2. After the panning of the libraries with an antigen off-rate-based selection, a number of active clones, most of which showed significantly improved affinity and specificity, were isolated, characterized and sequenced. The results indicate that the structure of the antibody can tolerate a number of different insertions in the CDRH2 region. They also suggest that the repertoire of antibody libraries can be expanded by extending the length of the CDR loops beyond that naturally provided by the given set of germline genes. This kind of mutagenesis can be generally useful for the engineering of hapten-binding antibodies.
Asunto(s)
Estradiol/inmunología , Región Variable de Inmunoglobulina/química , Región Variable de Inmunoglobulina/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión de Anticuerpos/genética , Cisteína/química , Cisteína/genética , Cartilla de ADN/genética , Haptenos/inmunología , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/genética , Técnicas In Vitro , Cinética , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Insercional , Conformación Proteica , Ingeniería de Proteínas , Esteroides/químicaRESUMEN
We have cloned and sequenced the first nonmammalian CD4 cDNA from the chicken using the COS cell expression method. Chicken CD4 contains four extracellular Ig domains that, in analogy to mammalian CD4, are in the order V, C2, V, and C2. The molecule is 24% identical with both human and mouse sequences. The extracellular domains were modeled using human and rat CD4 crystal structures as templates. In the first domain there are two extra Cys residues that are at suitable distance to form an intra-beta-sheet disulfide bridge in addition to the canonical one in the V domain. The region responsible for the interaction with MHC class II is relatively nonconserved in chicken. However, there are positively charged amino acids in the C" region of the N-terminal domain that may mediate the association to the negatively charged residues of the MHC class II beta-chain. Molecular modeling also implies that the membrane-proximal domain mediates dimerization of chicken CD4 in a similar way as it does for human CD4. Furthermore, the cytoplasmic tail is highly conserved, containing the protein tyrosine kinase p56lck recognition site that is preceded by an adjacent di-leucine motif for the internalization of the molecule. Interestingly, there are no Ser residues in the cytoplasmic part, which may explain the slow down-regulation of chicken CD4 after phorbol ester stimulation.
Asunto(s)
Antígenos CD4/química , Antígenos CD4/genética , Pollos/genética , Pollos/inmunología , Modelos Moleculares , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/química , Especificidad de Anticuerpos , Secuencia de Bases , Antígenos CD4/metabolismo , Línea Celular , Clonación Molecular , Citoplasma/inmunología , Citoplasma/metabolismo , ADN Complementario/inmunología , ADN Complementario/aislamiento & purificación , Humanos , Datos de Secuencia Molecular , Linfocitos T/metabolismoRESUMEN
An anti-estradiol antibody with improved specificity is searched for by combining steroid analog binding studies, mutant antibodies obtained from a phage-display library and structural modeling. Three-dimensional models for the anti-estradiol antibody 57-2 were constructed by comparative model building. Estradiol and analogs were docked into the combining site and molecular dynamics simulation was used to further refine this area of the protein. Cross-reactivities measured against 36 steroid analogs were used to help in the docking process and to evaluate the models. The roles of a number of residues were assessed by characterization of cross-reactivity mutants obtained from a phage display library. The cross-reactivity data and the results observed for mutants are explained by the structural model, in which the estradiol D-ring inserts deeply into the binding site and interacts with the antibody through at least one specific hydrogen bond. The binding data strongly suggest that this hydrogen bond connects the estradiol 17-hydroxyl group with the side chain of Gln H35. As expected for the binding of a small aromatic molecule, the antibody binding site contains many aromatic residues, e.g. Trp H50, H95 and L96 and Tyr L32, L49 and Phe L91.
Asunto(s)
Anticuerpos/inmunología , Especificidad de Anticuerpos , Estradiol/inmunología , Modelos Moleculares , Secuencia de Aminoácidos , Animales , Anticuerpos/química , Anticuerpos/genética , Genes de Inmunoglobulinas , Humanos , Ratones , Datos de Secuencia Molecular , Ingeniería de Proteínas , Análisis de Secuencia , Relación Estructura-ActividadRESUMEN
Bacillus stearothermophilus neopullulanase (NPL) structure was modeled based on Aspergillus oryzae alpha-amylase (TAA) to understand the structure-function relationships of this pullulan hydrolyzing enzyme. The NPL structure seems to consist of a central (alpha/beta)8 barrel to which the other domains are attached. The immediate surroundings of the NPL catalytic site were found to have very similar structure to TAA. The more distant sites are different due to the stereochemical requirements of accommodating in the substrate alpha-1,6-linkages at every third position instead of alpha-1,4-linkages. The substrate binding cleft is wider than in alpha-amylases. The NPL structure, function, substrate binding and the consequences of mutations were discussed based on the modeled structure.
Asunto(s)
Geobacillus stearothermophilus/enzimología , Glicósido Hidrolasas/química , Glicósido Hidrolasas/genética , Mutagénesis , Secuencia de Aminoácidos , Aspergillus oryzae/enzimología , Sitios de Unión , Secuencia Conservada , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Estructura Secundaria de Proteína , Alineación de Secuencia , Relación Estructura-Actividad , alfa-Amilasas/química , alfa-Amilasas/genéticaRESUMEN
Oligonucleotides labelled with detectable groups are essential tools in gene detection. We describe here the synthesis of pyrimidine deoxynucleotide-building blocks, modified at their C-5 position with a protected form of a strongly chelating agent. These reagents can be used to introduce multiple metal ions into oligodeoxynucleotides during standard oligonucleotide synthesis. The chelating functions form strongly fluorescent complexes with europium ions, characterized by a wide separation between the excitation and emission spectra. Moreover, the long decay time of the fluorescence permits sensitive time-resolved fluorescence measurements. The chelates also have the stability required to function in triple-color assays involving europium, samarium, and terbium ions. We demonstrate the application of these reagents for ligase-based gene analysis reactions.