RESUMO
The production of recombinant proteins is an essential tool for the expansion of modern biological research and biotechnology. The expression of heterologous proteins in Escherichia coli often results in an incomplete folding process that leads to the accumulation of inclusion bodies (IB), aggregates that hold a certain degree of native-like secondary structure. High hydrostatic pressure (HHP) impairs intermolecular hydrophobic and electrostatic interactions, leading to dissociation of aggregates under non-denaturing conditions and is therefore a useful tool to solubilize proteins for posterior refolding. Cholera toxin (CT) is composed of a non-toxic pentamer of B subunits (CTB), a useful adjuvant in vaccines, and a toxic subunit A (CTA). We studied the process of refolding of CTB using HHP. HHP was shown to be effective for dissociation of CTB monomers from IB. Posterior incubation at atmospheric pressure of concentrated CTB (1mg/ml) is necessary for the association of the monomers. Pentameric CTB was obtained when suspensions of CTB IB were compressed at 2.4kbar for 16h in the presence of Tween 20 and incubated at 1bar for 120h. Soluble and biologically active pentameric CTB was obtained, with a yield of 213mg CTB/liter of culture. The experience gained in this study can be important to improve the refolding of proteins with quaternary structure.
Assuntos
Toxina da Cólera/química , Toxina da Cólera/metabolismo , Redobramento de Proteína , Vibrio cholerae/genética , Toxina da Cólera/genética , Dicroísmo Circular , Escherichia coli/metabolismo , Pressão Hidrostática/efeitos adversos , Modelos Moleculares , Conformação Proteica , Multimerização Proteica , Estrutura Quaternária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Vibrio cholerae/metabolismoRESUMO
Replication and transcription of the human respiratory syncytial virus (hRSV) genome is carried out by the ribonucleocapsid complex (RNA together with N, P, M2-1 and L proteins), with the L protein being responsible for all enzymatic activities. In the present study, we obtained anti-L polyclonal sera in mice. These antibodies were functional in immunofluorescence and Western blotting assays in hRSV-infected HEp-2 cells. In the immunofluorescence assays, we detected inclusion bodies in the anti-L staining, similar to the ones seen by anti-N or anti-P staining. The results presented here provide the first evidence of the intracellular localization of the hRSV L protein.
