RESUMEN
The cDNA encoding an isoform of the cypress major pollen allergen, Cup a1.02, has been cloned and expressed in Escherichia coli as a N-terminal 6x His-tagged protein. To increase recovery, Cup a1.02 was expressed at high levels exploiting the T5 strong promoter and led to accumulate as inclusion bodies. The insoluble purified aggregates were solubilized in 6 M guanidine hydrochloride, immobilized using nickel-chelating affinity chromatography, and successfully refolded by controlled removal of the chaotropic reagent. Enhanced protein refolding was observed by reducing the protein concentration at 0.6-0.8 mg/ml. SDS-PAGE and gel filtration chromatography indicated an apparent molecular mass of approximately 40 kDa and the occurrence of the protein as monomers. The reconstituted fusion protein displayed the same immunological properties of the native Cup a1.02 protein as proven by IgE immunoreactivity. Immunoblotting, ELISA, and histamine release test showed that the tag did not preclude the protein functionality hence validating its correct three-dimensional folding. The protein fold was also assessed by CD spectroscopy and deconvolution of the spectrum allowed to estimate the secondary structure as a prevalence of beta structures (higher than 60%) and a small contribution from alpha helices (less than 12%). The reported procedure has proven to be useful for the production of multi-milligrams of recombinant Cup a1.02 allergen suitable for structural biology studies and for the molecular and functional characterization of the IgE binding sites.
Asunto(s)
Alérgenos/química , Cupressus/metabolismo , Escherichia coli/metabolismo , Proteínas de Plantas/química , Antígenos de Plantas , Sitios de Unión , Western Blotting , Cromatografía , Clonación Molecular , ADN Complementario/metabolismo , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Histamina/química , Humanos , Immunoblotting , Inmunoglobulina E/química , Modelos Biológicos , Níquel/química , Polen/metabolismo , Desnaturalización Proteica , Pliegue de Proteína , Isoformas de Proteínas , Estructura Terciaria de Proteína , Proteínas Recombinantes/químicaRESUMEN
Spermine oxidase (SMO) is a recently described flavoenzyme belonging to the class of polyamine oxidases (PAOs) and participating in the polyamine metabolism in animal cells. In this paper we describe the expression, purification, and characterization of the catalytic properties of a recombinant mouse SMO (mSMO). The purified enzyme has absorbance peaks at 457nm (epsilon=11mM(-1)cm(-1)) and 378nm, shows a molecular mass of approximately 63kDa, and has K(m) and k(cat) values of 170microM and 4.8s(-1), using spermine as substrate; it is unable to oxidize other free or acetylated polyamines. The mechanism-based PAO inhibitor N,N(1)-bis(2,3-butadienyl)-1,4-butanediamine (MDL72,527) acts as a competitive inhibitor of mSMO, with an apparent dissociation constant K(i)=63microM. If incubated for longer times, MDL72,527 yields irreversible inhibition of the enzyme with a half-life of 15min at 100microM MDL72,527. The mMSO catalytic mechanism, investigated by stopped flow, is consistent with a simple four-step kinetic scheme.
Asunto(s)
Escherichia coli/enzimología , Modelos Químicos , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/química , Putrescina/análogos & derivados , Putrescina/química , Animales , Catálisis , Simulación por Computador , Activación Enzimática , Inhibidores Enzimáticos/química , Estabilidad de Enzimas , Escherichia coli/genética , Isoenzimas , Cinética , Ratones , Oxidación-Reducción , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/antagonistas & inhibidores , Ingeniería de Proteínas , Especificidad por Sustrato , Poliamino OxidasaRESUMEN
Grass pea seedling histaminase (a copper-diamine oxidase) was found to exert a significant cardioprotection against post-ischaemic reperfusion damage. Electrocardiogram (ECG) recordings from the rats subjected in vivo to ischaemia and reperfusion showed ventricular tachycardia (VT) and ventricular fibrillations (VF) occurring in 9 out of 12 untreated rats whereas no ventricular arrhythmias were found under histaminase (80U/kg body weight) treatment (n=16 rats). Computer-assisted morphometry of the ischaemic reperfused hearts stained with nitroblue tetrazolium showed the extension of damaged myocardium (area at risk and infarct size) significantly reduced in rats treated with histaminase, in comparison with the non-treated rats, whereas no protection was found with the semicarbazide inactivated histaminase. Biochemical markers of ischaemia-reperfusion myocardial tissue damage: malonyldialdehyde (MDA), tissue calcium concentration, myeloperoxidase (MPO), and apoptosis indicator caspase-3 were significantly elevated in untreated post-ischaemic reperfused rats, but significantly reduced under histaminase protection. In conclusion, plant histaminase appears to protect hearts from ischaemia-reperfusion injury by more than one mechanism, essentially involving histamine oxidation, and possibly as reactive oxygen species scavenger, presenting good perspectives for a novel therapeutic approach in treatment of ischaemic heart pathology.