RESUMEN
The virus removal of protein A affinity chromatography, inactivation capacity, acid pH and a combination of high temperature with a chaotropic agent was determined in this work. The model viruses studied were sendaivirus, human immunodeficency virus (HIV-IIIb), human poliovirus type-II, human herpesvirus I and canine parvovirus. The protein A affinity chromatography showed a maximum reduction factor of 8 logs in the case of viruses larger than 120 nm size, while for small viruses (18-30 nm) the maximum reduction factor was about 5 logs. Non viral inactivation was observed during the monoclonal antibody elution step. Low pH treatment showed a maximum inactivation factor of 7.1 logs for enveloped viruses. However, a weak inactivation factor (3.4 logs) was obtained for DNA nonenveloped viruses. The combination of high temperature with 3 M KSCN showed a high inactivation factor for all of the viruses studied. The total clearance factor was 23.1, 15.1, 13.6, 20.0 and 16.0 logs for sendaivirus, HIV-IIIb, human poliovirus type-II, human herpesvirus I and canine parvovirus, respectively.
Asunto(s)
Cromatografía de Afinidad/métodos , Antígenos de Superficie de la Hepatitis B/inmunología , Vacunas de Productos Inactivados , Vacunas Sintéticas , Virus/aislamiento & purificación , Animales , Anticuerpos Monoclonales/análisis , Perros , Contaminación de Medicamentos/prevención & control , Estudios de Factibilidad , VIH/inmunología , VIH/aislamiento & purificación , Vacunas contra Hepatitis B/biosíntesis , Calor , Humanos , Concentración de Iones de Hidrógeno , Ratones , Ratones Endogámicos BALB C , Parvovirus/inmunología , Parvovirus/aislamiento & purificación , Poliovirus/inmunología , Poliovirus/aislamiento & purificación , Virus Sendai/inmunología , Virus Sendai/aislamiento & purificación , Sensibilidad y Especificidad , Proteína Estafilocócica A/química , Proteína Estafilocócica A/inmunología , Virus/inmunologíaRESUMEN
Recombinant human interferon alpha2b was expressed intracellularly in Escherichia coli as insoluble aggregates using a new expression vector, and was purified to homogeneity using essentially two-step chromatographic procedures, i.e. immobilized metal-ion-affinity chromatography and reversed-phase HPLC. The established purification process is highly reproducible and leads to a total recovery of approx. 12% with a specific biological activity of higher than 1x10(8) i.u./mg of protein, which is comparable with the international requirement for interferon alpha2b. For purified protein we report conformational stability as a function of pH and temperature using differential scanning calorimetry and CD. Thermal unfolding as a function of pH showed only one endotherm at a temperature higher than 45 degrees C, and was reversible at pH 2-3.75 and irreversible at pH 4-10. At pH 7.0, the most stable condition, the conformational stability depends on protein concentration and ionic strength. The highly helical secondary structure is very conserved over the whole pH range studied, including at high temperatures.
Asunto(s)
Escherichia coli/genética , Interferón-alfa/química , Interferón-alfa/aislamiento & purificación , Rastreo Diferencial de Calorimetría/métodos , Cromatografía en Agarosa/métodos , Cromatografía Líquida de Alta Presión/métodos , Dicroismo Circular , Humanos , Interferón alfa-2 , Interferón-alfa/genética , Concentración Osmolar , Conformación Proteica , Ingeniería de Proteínas/normas , Proteínas Recombinantes , Reproducibilidad de los ResultadosRESUMEN
The temperature dependence of the heat capacity function of a recombinant streptokinase (rSK) has been studied by high-sensitivity differential scanning microcalorimetry and circular dichroism as a function of pH in low- and high-ionic strength buffers. At low ionic strength it is found that this protein, between pH 7 and 10, undergoes four reversible and independent two-state transitions during its unfolding, suggesting the existence of four domains in the native structure of the protein. This result reconciles previous conflicting reports about the number of domains of this protein obtained by differential scanning calorimetry and small-angle X-ray scattering. The number of two-state transitions decreases when the pH of the medium is decreased, without noticeable changes in its circular dichroism spectrum. A plausible localization of the four domains in the streptokinase sequences is proposed and their thermodynamic parameters are given. Increase of ionic strength to 200 mM NaCl affects positively the protein stability and confirms the existence of four reversible two-state transitions. Above 200 mM NaCl the protein stability decreases, resulting in low percentage of reversibility, and even irreversible transitions.
Asunto(s)
Rastreo Diferencial de Calorimetría , Estreptoquinasa/química , Dicroismo Circular , Estabilidad de Enzimas , Concentración de Iones de Hidrógeno , Mutación , Conformación Proteica , Desnaturalización Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Cloruro de Sodio/química , Estreptoquinasa/genética , Estreptoquinasa/metabolismoRESUMEN
We studied, for the first time, characterization of the invertase expressed in the methylotrophic yeasts Hansenula polymorpha and Pichia pastoris in terms of enzyme conformational stability and structural behaviour induced by temperature as a function of pH using enzymic assays, differential scanning calorimetry, fluorescence and CD. The enzyme produced in both hosts was very stable over a broad range of pH values, keeping its enzymic activity and structure above 60 degrees C. Thermal denaturation, as measured by differential scanning calorimetry, was always irreversible. However, the fact that scanning rate had no effect on the calorimetric curves gave us the chance to analyse the data from a thermodynamic point of view. The conformational stabilities were essentially identical under the experimental conditions studied, but stability was always slightly higher in the enzyme expressed in H. polymorpha. This fact indicates that the greater degree of glycosylation of this enzyme form contributed to its increased global stability. Reactivation upon heating at 80 degrees C depends on protein concentration, suggesting that irreversibility could be associated with slow refolding kinetics at high protein concentration.
Asunto(s)
Glicósido Hidrolasas/química , Glicósido Hidrolasas/metabolismo , Pichia/genética , Rastreo Diferencial de Calorimetría , Dicroismo Circular , Estabilidad de Enzimas , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/aislamiento & purificación , Glicosilación , Concentración de Iones de Hidrógeno , Cinética , Conformación Proteica , Desnaturalización Proteica , Pliegue de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Espectrometría de Fluorescencia , Temperatura , beta-FructofuranosidasaRESUMEN
The aspartic proteinase gene of Mucor pusillus rennin expressed in Pichia pastoris was characterized in terms of structural and conformational stability induced by temperature. This enzyme is 12% glycosylated, with a similar specific activity to the native fungal enzyme. The secondary structure determined by CD is mainly due to beta-sheet structures with an important contribution of aromatic components. The calorimetric studies were carried out in the temperature range in which the enzyme is most stable. The enzyme undergoes an irreversible, highly scan-rate-dependent thermal denaturation under all the experimental conditions investigated. Between pH 3.0 and 7.0, only one endotherm characterized the thermal denaturation of enzyme. At pH 5.0, the most stable condition found, the denaturation can be fitted to the two-state irreversible model. Thus the kinetic constant and activation parameters of the denaturation process could be obtained. Upon reaching pH 7.5, the denaturation is characterized by two endotherms. This evidence indicates the complex tridimensional structure of this enzyme. Finally, taking into account the conservative tertiary structure of the aspartic proteinase family we comment on our results with reference to the crystallographic structure of M. pusillus pepsin [Newman, Watson, Roychowdhury, Badasso, Cleasby, Wood, Tickle and Blundell (1993) J. Mol. Biol. 221, 1295-1309].
Asunto(s)
Quimosina/química , Mucor/enzimología , Biotecnología , Rastreo Diferencial de Calorimetría , Quimosina/genética , Quimosina/metabolismo , Dicroismo Circular , Estabilidad de Enzimas , Expresión Génica , Genes Fúngicos , Glicosilación , Concentración de Iones de Hidrógeno , Peso Molecular , Mucor/genética , Pichia/genética , Conformación Proteica , Desnaturalización Proteica , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometría de Fluorescencia , TemperaturaRESUMEN
The thermal denaturation of a recombinant human gamma-interferon has been studied as a function of pH in the range from 2 to 10 and buffer concentration in the range from 5 to 100 mM by differential scanning calorimetry, circular dichroism, fluorescence, 1H NMR, and biological activity measurements. The thermal transitions are irreversible at high buffer concentrations at all pH values studied, although they are reversible between pH 3.5 and 5.4 at low buffer concentrations. The denaturation enthalpy, DeltaH(Tm), at denaturation temperature Tm was a function of both Tm and the buffer concentration, and this resulted in heat capacity changes decreasing with buffer concentration. When the denaturation enthalpies were corrected for Tm dependence, they did not appear to change versus pH. The denaturation entropies, however, appeared to decrease with pH, leading to a small but appreciable increase in the stability of the protein with pH. The difference between the number of moles of protons stoichiometrically bound to a mole of protein in the native and thermally denatured state, was calculated from the variation of Tm versus pH at each buffer concentration. The values obtained appear to depend on pH alone rather than upon temperature or buffer concentration, a result which agrees with the invariance of the denaturation enthalpies with pH. This dependence was fitted to the titration curve of a group with a pK of 5.4.
Asunto(s)
Interferón gamma/química , Interferón gamma/metabolismo , Rastreo Diferencial de Calorimetría/métodos , Dicroismo Circular , Deuterio , Calor , Humanos , Concentración de Iones de Hidrógeno , Interferón gamma/genética , Resonancia Magnética Nuclear Biomolecular , Desnaturalización Proteica , Pliegue de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espectrometría de Fluorescencia , TermodinámicaRESUMEN
A structural model is presented for family 32 of the glycosyl-hydrolase enzymes based on the beta-propeller fold. The model is derived from the common prediction of two different threading methods, TOPITS and THREADER. In addition, we used a correlated mutation analysis and prediction of active-site residues to corroborate the proposed model. Physical techniques (circular dichroism and differential scanning calorimetry) confirmed two aspects of the prediction, the proposed all-beta fold and the multi-domain structure. The most reliable three-dimensional model was obtained using the structure of neuraminidase (1nscA) as template. The analysis of the position of the active site residues in this model is compatible with the catalytic mechanism proposed by Reddy and Maley (J. Biol. Chem. 271:13953-13958, 1996), which includes three conserved residues, Asp, Glu, and Cys. Based on this analysis, we propose the participation of one more conserved residue (Asp 162) in the catalytic mechanism. The model will facilitate further studies of the physical and biochemical characteristics of family 32 of the glycosyl-hydrolases.
Asunto(s)
Proteínas Bacterianas , Glicósido Hidrolasas/química , Hexosiltransferasas/química , Conformación Proteica , Secuencia de Aminoácidos , Dicroismo Circular , Datos de Secuencia Molecular , Mutación , Filogenia , Saccharomyces cerevisiae/enzimología , beta-FructofuranosidasaRESUMEN
The DEX gene encodes an extracellular dextranase (EC 3.2.1.11); this enzyme hydrolyzes the alpha(1,6) glucosidic bond contained in dextran to release small isomaltosaccharides. Sequence analysis has revealed only one homologous sequence, CB-8 protein, from Arthrobacter sp., with 30% sequence identity. The secondary structure prediction for Dex was corroborated by circular dichroism measurements. To explore the possibility that Dex protein might adopt a fold similar to any known structure, we conducted a threading search of a three-dimensional structure database. This search revealed that the Dex sequence is compatible with the galactose oxidase/methanol dehydrogenase/sialidase fold. A structural model of Dex based on these results is physically and biologically plausible and leads to testable predictions, including the prediction that Asp246 and Glu299 might be catalytic residues. Also, according to this model the Dex enzyme has a mechanism of hydrolysis with net inversion of anomeric configuration.