RESUMEN
Glutathione peroxidase (GPX) is one of the important members of the antioxidant enzyme family. It can catalyze the reduction of hydroperoxides with glutathione to protect cells against oxidative damage. In previous studies, we have prepared the human catalytic antibody Se-scFv-B3 (selenium-containing single-chain Fv fragment of clone B3) with GPX activity by incorporating a catalytic group Sec (selenocysteine) into the binding site using chemical mutation; however, its activity was not very satisfying. In order to try to improve its GPX activity, structural analysis of the scFv-B3 was carried out. A three-dimensional (3D) structure of scFv-B3 was constructed by means of homology modeling and binding site analysis was carried out. Computer-aided docking and energy minimization (EM) calculations of the antibody-GSH (glutathione) complex were also performed. From these simulations, Ala44 and Ala180 in the candidate binding sites were chosen to be mutated to serines respectively, which can be subsequently converted into the catalytic Sec group. The two mutated protein and wild type of the scFv were all expressed in soluble form in Escherichia coli Rosetta and purified by Ni(2+)-immobilized metal affinity chromatography (IMAC), then transformed to selenium-containing catalytic antibody with GPX activity by chemical modification of the reactive serine residues. The GPX activity of the mutated catalytic antibody Se-scFv-B3-A180S was significantly increased compared to the original Se-scFv-B3.
Asunto(s)
Anticuerpos Catalíticos/química , Anticuerpos Catalíticos/metabolismo , Glutatión Peroxidasa/metabolismo , Región Variable de Inmunoglobulina/metabolismo , Mutagénesis Sitio-Dirigida , Mutación/genética , Selenio/metabolismo , Secuencia de Aminoácidos , Anticuerpos Catalíticos/genética , Anticuerpos Catalíticos/aislamiento & purificación , Sitios de Unión , Western Blotting , Células Clonales , Biología Computacional , Electroforesis en Gel de Poliacrilamida , Glutatión/química , Glutatión Peroxidasa/química , Glutatión Peroxidasa/genética , Humanos , Enlace de Hidrógeno , Región Variable de Inmunoglobulina/química , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/aislamiento & purificación , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Selenocisteína/metabolismo , Alineación de Secuencia , TermodinámicaRESUMEN
Production of anti-alphaIIbbeta3 (anti-alphaIIbbeta3)-binding single-chain FV (scFv) fragments obtained from combinatorial libraries of IgG human antibodies is of broad interest for imaging and treatment of acute coronary syndromes. The objective of our work was to design an optimized production of one selected anti-alphaIIbbeta3-binding scFv fragment for subsequent in vivo animal studies. Fed-batch fermentation was initiated with 2TY media supplemented with 0.1 M glucose. This growing batch culture was used as a starting point for further fed-batch induction, in which a media without glucose containing 1 mM IPTG and 0.4 M saccharose was continuously added. Subsequent purification was performed on the whole cell extract in native conditions over an immobilized copper-ion affinity column. The improved conditions allowed the recovery of 5 mg of highly purified scFv fragments as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The bioactivity of the scFv fragments was further monitored by ELISA, cytometric and immunohistochemical methods.
Asunto(s)
Cromatografía de Afinidad/métodos , Fragmentos de Inmunoglobulinas/aislamiento & purificación , Región Variable de Inmunoglobulina/aislamiento & purificación , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/inmunología , Animales , Plaquetas/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/metabolismo , Fermentación , Citometría de Flujo , Inmunohistoquímica , Masculino , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/farmacología , Conejos , Trombosis/inmunología , TransfecciónRESUMEN
We constructed a plasmid containing a chimeric gene composed of the gene encoding acetylcholinesterase (AChE) from Bungarus fasciatus venom and a gene encoding a single chain antibody fragment (scFv) directed against one of the two subunits of a presynaptic neurotoxin from rattlesnake. Large quantities of the fusion protein were produced in the culture medium of transfected COS cells. Fusion to AChE did not affect the ability of the scFv to recognise its antigen. Similarly, the AChE activity was not impaired in the fusion. The fusion protein was purified from the culture medium in a single step by affinity chromatography. The immunoconjugate obtained consisted of a soluble monomeric form of AChE fused to scFv. It was monovalent and had a molecular weight of 94 kDa. The properties of this scFv-AChE fusion show that the simple, reproducible preparation of various recombinant monovalent immunoenzymatic tracers with low molecular weight is possible. In addition, in the construct presented, the scFv domain can be easily changed to another one taking advantage of the SfiI-NotI restriction sites surrounding this domain.
Asunto(s)
Acetilcolinesterasa/genética , Venenos Elapídicos/enzimología , Región Variable de Inmunoglobulina/genética , Recombinación Genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bungarus , Células COS , Clonación Molecular , ADN Complementario , Electroforesis en Gel de Poliacrilamida , Técnicas para Inmunoenzimas , Región Variable de Inmunoglobulina/aislamiento & purificación , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificaciónRESUMEN
An autoantibody produced by a hybridoma derived from a viable motheaten mouse was isolated and found to have moderately high binding affinity for nucleic acids and specific preference for quadruplex DNAs. Polymerase chain reaction primers were designed to link the cloned parental antibody variable region fragments together in a subcloning vector. This single-chain variable fragment construct was then subcloned into the T7 promoter-driven expression vector pET22b(+). The construct contains (N- to C-terminal) a pelB leader sequence, variable heavy chain, glycine-serine polylinker, variable light chain, and biotin mimic peptide "strep-tag" sequence (pelB-VH-linker-VL-strep-tag). The ca. 29 kDa protein was expressed, exported to the periplasmic space of NovaBlue (DE) Escherichia coli, and purified by streptavidin affinity chromatography by binding the fused strep-tag peptide. The specificity of the purified single-chain variable fragment (scFv) for quadruplex and duplex DNAs was evaluated by a radioimmunofilterbinding assay. It retained about 10-fold higher affinity for quadruplexes relative to duplex DNA, a reduction of ca. 4-fold from the relative preferences of the parent IgG. The complementary-determining regions contain sequences that are homologous to or conservatively divergent from the key DNA-binding helix-turn-helix-forming motifs of Myb/RAP1 family telomeric DNA-binding proteins (1-3). The presence of this antibody in the autoimmune repertoire suggests a possible linkage between autoimmunity, telomeric DNA binding proteins, and aging.
Asunto(s)
Anticuerpos Antinucleares/química , Proteínas de Unión al ADN/química , ADN/inmunología , Hibridomas/química , Región Variable de Inmunoglobulina/química , Homología de Secuencia de Aminoácido , Secuencia de Aminoácidos , Animales , Anticuerpos Antinucleares/genética , Anticuerpos Antinucleares/aislamiento & purificación , Secuencia de Bases , Clonación Molecular , ADN/metabolismo , G-Cuádruplex , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/aislamiento & purificación , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Ingeniería de Proteínas/métodos , Proteínas Recombinantes de Fusión/síntesis química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Anticuerpos de Cadena Única , Telómero/metabolismoRESUMEN
By establishing hybridomas from two distinct surface IgM+ splenic B cell populations, Ly-1 B cells and "conventional" (Ly-1-) B cells, we found that the Ly-1 B population includes a 30 to 70 times higher frequency (1 to 2%) of cells with specificity for bromelain treated autologous red blood cells (anti-BrMRBC) when compared with conventional B cells (0.03%). We cloned and sequenced the V genes encoding anti-BrMRBC antibody from two hybridomas made with Ly-1 B cells sorted from the spleen of SM/J mice. The VH sequence (for both) is identical with the previously reported sequence associated with this specificity and belongs to a new VH gene family. This gene family, defined here as VH11, has only two members and is the predominant VH rearranged in a collection of Ly-1 B derived anti-BrMRBC hybridomas, always in association with a single VL gene (a member of the V kappa 9 family). Furthermore, analysis of hybridomas made with Ly-1 B cells sorted from the peritoneum reveals a yet higher increased frequency of VH11-encoded anti-BrMRBC specificity (30%). This variation in frequency of anti-BrMRBC in the Ly-1 population depending on location, together with the repeated association of VH11 with a particular V kappa gene suggest that antigen driven selection is (at least in part) responsible for the biased V gene expression seen in this population. Furthermore, a mechanism that might contribute to biased expression, preferential rearrangement due to close proximity to J (as seen in pre-B lines), is excluded by localization of VH11 5' to several of the more J-proximal families (Q52, 7183).