RESUMO
Actin is the most abundant protein in the cytoplasm of most eukaryotic cells and is involved in a variety of cellular functions. It has been difficult to produce actin in bacterial expression systems in good yields. In this study, we developed a new simple method for the production of recombinant actin in Escherichia coli cells. Human ß-actin was successfully expressed using a cold shock vector, pCold, in the bacterial expression system. The expressed ß-actin (hexahistidine-tagged) was separated with a Ni-chelating resin, followed by a polymerization/depolymerization cycle or column chromatography with the Ni-chelating resin. The purified recombinant ß-actin showed a normal polymerization ability compared with ß-actin purified from human platelets. We produced a recombinant mutant actin with a Gly-168Arg mutation in the system and confirmed that it exhibited an impaired polymerization ability. The system developed in this study will provide a useful method for the production of actin isoforms and their mutants.
Assuntos
Actinas/biossíntese , Proteínas e Peptídeos de Choque Frio/genética , Proteínas Recombinantes de Fusão/biossíntese , Actinas/genética , Actinas/isolamento & purificação , Actinas/metabolismo , Biotecnologia/métodos , Western Blotting , Cromatografia de Afinidade , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Vetores Genéticos/genética , Histidina/química , Humanos , Mutação , Oligopeptídeos/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificaçãoRESUMO
p40(phox) activated phagocyte NADPH oxidase without p47(phox) in a cell-free system consisting of p67(phox), Rac and cytochrome b(558) relipidated with phosphatidylinositol 3-phosphate. The activation reached to 70% of that by p47(phox). Addition of p47(phox) slightly increased the activation, but not additively. p40(phox) improved the efficiency of p67(phox) in the activation. The C-terminus-truncated p67(phox), p40(phox)(D289A), p40(phox)(R58A), or p40(phox)(W207R) showed an impaired activation. A peptide corresponding to the p22(phox) Pro-rich region suppressed the activation, and far-western blotting revealed its interaction with p40(phox) SH3 domain. Thus, p40(phox) can substitute for p47(phox) in the activation, interacting with p22(phox) and p67(phox) through their specific regions.
Assuntos
NADPH Oxidases/metabolismo , Fosfoproteínas/metabolismo , Sequência de Aminoácidos , Animais , Far-Western Blotting , Bovinos , Sistema Livre de Células , Ativação Enzimática/efeitos dos fármacos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Dados de Sequência Molecular , Mutação , NADPH Oxidases/genética , Fosfoproteínas/genética , Ligação Proteica , Homologia de Sequência de Aminoácidos , Dodecilsulfato de Sódio/farmacologia , Domínios de Homologia de srcRESUMO
The effects of reactive oxygen species on cells have attracted great attention from both physiological and pathological aspects. Superoxide (O2-) is the primary reactive oxygen species formed in animals. We previously developed an O2--generating nanodevice consisting of NADPH oxidase 2 (Nox2) and modulated activating factors. However, the device was subsequently found to be unstable in a standard culture medium. Here we improved the device in stability by cross-linking. This new nanodevice, Device II, had a half-life of 3 h at 37 °C in the medium. Device II induced cell death in 80% of HEK293 cells after 24 h of incubation. Superoxide dismutase alone did not diminish the effect of the device, but eliminated the effect when used together with catalase, confirming that the cell death was caused by H2O2 derived from O2-. Flow cytometric analyses revealed that Device II induced caspase-3 activation in HEK293 cells, suggesting that the cell death proceeded largely through apoptosis.