RESUMO

This paper reports a theoretical and experimental investigation on the recombinant protein rotavirus VP6 as a bioelectrochemical interface. Our motivation arises from the highly active zones of VP6 which can interact with biological structures and metals, as well as its useful features such as self-assembly, polymorphism, and active surface charge. A molecular simulation study was performed to analyze the charge transfer properties of theVP6 trimer under an applied electric field. The electrostatic properties were evaluated via the nonlinear second-order Poisson-Boltzmann equation, using finite element methods based on parameter discretization and calculation of solute/solvent interaction forces, which account for mean-field screening effects. The electrochemical study validated the theoretical predictions for VP6 in their different assemblies (trimers and nanotubes) when they are used as electrodes in 10 mM K3[Fe(CN)6], 1 M KCl. Applying a potential sweep promotes charge transfer, facilitates redox activity of the ferricyanide ion. Furthermore, protein assemblies decreased electrode electrical resistance and enabled gold particle electrodeposition on the protein VP6. These results suggest that VP6 is a promising conductive biomaterial that promotes charge transfer of redox probes and could be used as a new scaffold to create bio-electrochemical interfaces.

Assuntos

Antígenos Virais/química , Proteínas do Capsídeo/química , Proteínas Imobilizadas/química , Nanotubos/química , Rotavirus/química , Condutividade Elétrica , Técnicas Eletroquímicas , Eletrodos , Polímeros de Fluorcarboneto/química , Modelos Moleculares , Multimerização Proteica , Proteínas Recombinantes/química , Eletricidade Estática

RESUMO

Latex from Hevea brasiliensis contains several allergenic proteins that are involved in type I allergy. One of them is Hev b 2, which is a beta-1,3-glucanase enzyme that exists in different isoforms with variable glycosylation content. Two glucanase isoforms were isolated from trees of the GV-42 clone by gel filtration, affinity and ion-exchange chromatography. Isoform I had a carbohydrate content of about 20%, with N-linked N-acetyl-glucosamine, N-acetyl-galactosamine, fucose and galactose residues as the main sugars, while isoform II showed 6% carbohydrate content consisting of N-acetyl-glucosamine, fucose, mannose and xylose. Both isoforms were crystallized by the hanging-drop vapour-diffusion method. Isoform I crystals were grown using 0.2 M trisodium citrate dihydrate, 0.1 M Na HEPES pH 7.5 and 20%(v/v) 2-propanol, but these crystals were not appropriate for data collection. Isoform II crystals were obtained under two conditions and X-ray diffraction data were collected from both. In the first condition (0.2 M trisodium citrate, 0.1 M sodium cacodylate pH 6.5, 30% 2-propanol), crystals belonging to the tetragonal space group P4(1) with unit-cell parameters a = b = 150.17, c = 77.41 A were obtained. In the second condition [0.2 M ammonium acetate, 0.1 M trisodium citrate dihydrate pH 5.6, 30%(w/v) polyethylene glycol 4000] the isoform II crystals belonged to the monoclinic space group P2(1), with unit-cell parameters a = 85.08, b = 89.67, c = 101.80 A, beta = 113.6 degrees. Preliminary analysis suggests that there are four molecules of isoform II in both asymmetric units.

Assuntos

Alérgenos/química , Proteínas de Plantas/química , Alérgenos/genética , Alérgenos/isolamento & purificação , Sequência de Aminoácidos , Antígenos de Plantas , Glicoproteínas/química , Glicoproteínas/isolamento & purificação , Hevea , Dados de Sequência Molecular , Monossacarídeos/análise , Fragmentos de Peptídeos/química , Proteínas de Plantas/genética , Proteínas de Plantas/isolamento & purificação , Polimorfismo Genético , Isoformas de Proteínas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Difração de Raios X

RESUMO

High productivities of bioprocesses involving viruses can be attained through infection strategies based on adequate understanding of parameters ruling cell-virus interactions. Two factors that affect virus binding and infection efficiency were studied: the utilization of an adsorption step, where infection volume at constant cell/virus ratio was varied; and the concentration of fetal bovine serum (FBS). The insect cell-baculovirus expression system and recombinant protein VP4 of rotavirus were used as models. Virus binding kinetics were adequately described by a sigmoidal response curve. The adsorption step, with or without FBS, increased virus attachment rate, whereas it increased bound virus at equilibrium only in FBS-free infections. A first-order dependance of virus attachment on cell concentration was found above 5 x 10(6) cell/mL in infections with 10% FBS. Addition of 10% FBS decreased maximum bound baculovirus and binding rate by as much as 3 times and VP4 concentration up to 4 times. In contrast, heat inactivation of FBS increased bound virus from 20% to over 90%, an increase of 1.4 times compared to FBS-free infections. A direct linear relation was found between attached virus and maximum VP4 concentration for the different FBS concentrations tested, indicating that baculovirus-cell attachment was the limiting step for recombinant protein production. Interestingly, virus progeny accumulation was not affected by differences in virus binding. In conclusion, infection strategies aimed at increasing productivity should be performed at high cell concentrations and without FBS, or with heat-inactivated FBS.