RESUMEN
The detection and purification of proteins are often time-consuming and frequently involve complicated protocols. The addition of a peptide tag to recombinant proteins can make this process more efficient. Many of the commonly used tags, such as Flagtrade mark, Myc, HA and V5 are recognized by specific monoclonal antibodies and therefore, allow immunoaffinity-based purification. Enhancing the current scope of flexibility in using diverse peptide tags, we report here the development of a novel, short polypeptide tag (Tab2) for detection and purification of recombinant proteins. The Tab2 epitope corresponds to the NH2-terminal seven amino acid residues of human TGFalpha. A monoclonal anti-Tab2 antibody was raised and characterized. To investigate the potential of this peptide sequence as a novel tag for recombinant proteins, we expressed several different recombinant proteins containing this tag in E. coli, baculovirus, and mammalian cells. The data presented demonstrates the Tab2 tag-anti-Tab2 antibody combination is a reliable tool enabling specific Western blot detection, FACS analysis, and immunoprecipitation as well as non-denaturing protein affinity purification.
Asunto(s)
Cromatografía de Afinidad/métodos , Epítopos/química , Fragmentos de Péptidos/inmunología , Proteínas Recombinantes de Fusión/aislamiento & purificación , Factor de Crecimiento Transformador alfa/genética , Secuencias de Aminoácidos/genética , Animales , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos/inmunología , Células Cultivadas , Clonación Molecular , Ensayo de Inmunoadsorción Enzimática/métodos , Citometría de Flujo/métodos , Vectores Genéticos/síntesis química , Humanos , Inmunoprecipitación/métodos , Insectos , Fragmentos de Péptidos/genética , Fosfotransferasas/genética , Fosfotransferasas/aislamiento & purificación , Fosfotransferasas/metabolismo , Unión Proteica/inmunología , Estructura Terciaria de Proteína/genética , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/aislamiento & purificación , Receptores de Superficie Celular/metabolismo , Proteínas Recombinantes de Fusión/síntesis química , Proteínas Recombinantes de Fusión/inmunología , Factor de Crecimiento Transformador alfa/química , Factor de Crecimiento Transformador alfa/metabolismoRESUMEN
Prolyl-4-hydroxylase domain-containing enzymes (PHDs) mediate the oxygen-dependent regulation of the heterodimeric transcription factor hypoxia-inducible factor-1 (HIF-1). Under normoxic conditions, one of the subunits of HIF-1, HIF-1alpha, is hydroxylated on specific proline residues to target HIF-1alpha for degradation by the ubiquitin-proteasome pathway. Under hypoxic conditions, the hydroxylation by the PHDs is attenuated by lack of the oxygen substrate, allowing HIF-1 to accumulate, translocate to the nucleus, and mediate HIF-mediated gene transcription. In several mammalian species including humans, three PHDs have been identified. We report here the cloning of a full-length rat cDNA that is highly homologous to the human and murine PHD-1 enzymes and encodes a protein that is 416 amino acids long. Both cDNA and protein are widely expressed in rat tissues and cell types. We demonstrate that purified and crude baculovirus-expressed rat PHD-1 exhibits HIF-1alpha specific prolyl hydroxylase activity with similar substrate affinities and is comparable to human PHD-1 protein.
Asunto(s)
Clonación Molecular , Procolágeno-Prolina Dioxigenasa/química , Procolágeno-Prolina Dioxigenasa/genética , Secuencia de Aminoácidos , Animales , Humanos , Cinética , Ratones , Datos de Secuencia Molecular , Especificidad de Órganos/genética , Procolágeno-Prolina Dioxigenasa/biosíntesis , ARN Mensajero/metabolismo , Ratas , SpodopteraRESUMEN
There has been intense interest in the development of factor Xa inhibitors for the treatment of thrombotic diseases. Our laboratory has developed a series of novel non-amidine inhibitors of factor Xa. This paper presents two crystal structures of compounds from this series bound to factor Xa. The first structure is derived from the complex formed between factor Xa and compound 1. Compound 1 was the first non-amidine factor Xa inhibitor from our lab that had measurable potency in an in vitro assay of anticoagulant activity. The second compound, 2, has a molar affinity for factor Xa (K(iapp)) of 7 pM and good bioavailability. The two inhibitors bind in an L-shaped conformation with a chloroaromatic ring buried deeply in the S1 pocket. The opposite end of these compounds contains a basic substituent that extends into the S4 binding site. A chlorinated phenyl ring bridges the substituents in the S1 and S4 pockets via amide linkers. The overall conformation is similar to the previously published structures for amidine-based inhibitors complexed with factor Xa. However, there are significant differences in the interactions between the inhibitor and the protein at the atomic level. Most notably, there is no group that forms a salt bridge with the carboxylic acid at the base of the S1 pocket (Asp189). Each inhibitor forms only one well-defined hydrogen bond to the protein. There are no direct charge-charge interactions. The results indicate that electrostatic interactions play a secondary role in the binding of these potent inhibitors.
