RESUMO
We previously generated a highly thermostable triple variant of Moloney murine leukemia virus reverse transcriptase, MM3 (E286R/E302K/L435R), by introducing positive charges by site-directed mutagenesis at positions that have been implicated in the interaction with template-primer (Yasukawa et al., (2010) J. Biotechnol., 150, 299-306). In this study, we attempted to further increase the thermostability of MM3. Twenty-nine mutations were newly designed, focusing on the number of surface charge, stabilization of hydrophobic core, and introduction of salt bridge. The corresponding 29 single variants were produced in Escherichia coli and characterized for activity and stability. Six mutations (A32V, L41D, L72R, I212R, L272E and W388R) were selected as the candidates for further stabilize MM3. Fifteen multiple variants were designed by combining two or more of the six mutations with the MM3 mutations, produced and characterized. The sextuple variant MM3.14 (A32V/L72R/E286R/E302K/W388R/L435R) exhibited higher thermostability than MM3.
Assuntos
Vírus da Leucemia Murina de Moloney/genética , Mutagênese Sítio-Dirigida/métodos , DNA Polimerase Dirigida por RNA/genética , Proteínas Recombinantes/genética , Proteínas Virais/genética , Estabilidade Enzimática , Escherichia coli/genética , Temperatura Alta , Vírus da Leucemia Murina de Moloney/enzimologia , DNA Polimerase Dirigida por RNA/química , DNA Polimerase Dirigida por RNA/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismoRESUMO
We implemented a noninvasive optical method for the fast control of Cre recombinase in single cells of a live zebrafish embryo. Optical uncaging of the caged precursor of a nonendogeneous steroid by one- or two-photon illumination was used to restore Cre activity of the CreER(T2) fusion protein in specific target cells. This method labels single cells irreversibly by inducing recombination in an appropriate reporter transgenic animal and thereby can achieve high spatiotemporal resolution in the control of gene expression. This technique could be used more generally to investigate important physiological processes (e.g., in embryogenesis, organ regeneration, or carcinogenesis) with high spatiotemporal resolution (single cell and 10-min scales).
Assuntos
Regulação Enzimológica da Expressão Gênica/fisiologia , Integrases/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Recombinação Genética/fisiologia , Peixe-Zebra , Animais , Animais Geneticamente Modificados , Primers do DNA/genética , Proteínas de Choque Térmico HSP70/metabolismo , Microscopia de Fluorescência , Processos Fotoquímicos , Reação em Cadeia da Polimerase , Espectrometria de FluorescênciaRESUMO
The accumulation of PrP(Sc) in scrapie-infected neuronal cells has been prevented by three approaches: (i) transfection of ScMNB cells with an antisense laminin receptor precursor (LRP) RNA-expression plasmid, (ii) transfection of ScN2a cells and ScGT1 cells with small interfering RNAs (siRNAs) specific for the LRP mRNA, and (iii) incubation of ScN2a cells with an anti-LRP/LR antibody. LRP antisense RNA and LRP siRNAs reduced LRP/LR expression and inhibited the accumulation of PrP(Sc) in these cells. The treatments also reduced PrP(c) levels. The anti-LRP/LR antibody, W3, abolished PrP(Sc) accumulation and reduced PrP(c) levels after seven days of incubation. Cells remained free of PrP(Sc) after being cultured for 14 additional days without the antibody, whereas the PrP(c) level was restored. Our results demonstrate the necessity of the laminin receptor (LRP/LR) for PrP(Sc) propagation in cultured cells and suggest that LRP/LR-specific antibodies could be used as powerful therapeutic tools in the treatment of transmissible spongiform encephalopathies.
Assuntos
Neurônios/metabolismo , Proteínas PrPSc/metabolismo , Receptores de Laminina/metabolismo , Scrapie/metabolismo , Animais , Transporte Biológico , Peso Molecular , Neuroblastoma , Proteínas PrPSc/genética , RNA Antissenso/farmacologia , RNA Interferente Pequeno/farmacologia , Receptores de Laminina/química , Receptores de Laminina/genética , Receptores de Laminina/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribonucleases , Scrapie/genética , Células Tumorais CultivadasRESUMO
There is evidence that prion protein dimers may be involved in the formation of the scrapie prion protein, PrP(Sc), from its normal (cellular) form, PrP(c). Recently, the crystal structure of the human prion protein in a dimeric form was reported. Here we report for the first time the overexpression of a human PrP dimer covalently linked by a FLAG peptide (PrP::FLAG::PrP) in the methylotrophic yeast Pichia pastoris. FLAG-tagged human PrP (aa1-aa253) (huPrP::FLAG) was also expressed in the same system. Treatment with tunicamycin and endoglycosidase H showed that both fusion proteins are expressed as various glycoforms. Both PrP proteins were completely digested by proteinase K (PK), suggesting that the proteins do not have a PrP(Sc) structure and are not infectious. Plasma membrane fractionation revealed that both proteins are transported to the plasma membrane of the cell. The glycosylated proteins might act as powerful tools for crystallization trials, PrP(c)/PrP(Sc) conversion studies and other applications in the life cycle of prions.