RESUMO
We have previously shown that the small metal-binding protein (SmbP) extracted from the gram-negative bacterium Nitrosomonas europaea can be employed as a fusion protein for the expression and purification of recombinant proteins in Escherichia coli. With the goal of increasing the amounts of SmbP-tagged proteins produced in the E. coli periplasm, we replaced the native SmbP signal peptide with three different signal sequences: two were from the proteins CusF and PelB, for transport via the Sec pathway, and one was the signal peptide from TorA, for transport via the Tat pathway. Expression of SmbP-tagged Red Fluorescent Protein (RFP) using these three alternative signal peptides individually showed a considerable increase in protein levels in the periplasm of E. coli as compared to its level using the SmbP signal sequence. Therefore, for routine periplasmic expression and purification of recombinant proteins in E. coli, we highly recommend the use of the fusion proteins PelB-SmbP or CusF-SmbP, since these signal sequences increase periplasmic production considerably as compared to the wild-type. Our work, finally, demonstrates that periplasmic expression for SmbP-tagged proteins is not limited to the Sec pathway, in that the TorA-SmbP construct can export reasonable quantities of folded proteins to the periplasm. Although the Sec route has been the most widely used, sometimes, depending on the nature of the protein of interest, for example, if it contains cofactors, it is more appropriate to consider using the Tat route over the Sec. SmbP therefore can be recommended in terms of its particular versatility when combined with signal peptides for the two different routes.
Assuntos
Proteínas de Bactérias/genética , Clonagem Molecular/métodos , Nitrosomonas europaea/genética , Periplasma/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Proteínas de Transporte de Cobre , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Genes Reporter , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Proteínas de Ligação ao Ferro/genética , Proteínas de Ligação ao Ferro/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Nitrosomonas europaea/metabolismo , Oxirredutases N-Desmetilantes/genética , Oxirredutases N-Desmetilantes/metabolismo , Periplasma/química , Polissacarídeo-Liases/genética , Polissacarídeo-Liases/metabolismo , Sinais Direcionadores de Proteínas , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismo , Proteína Vermelha FluorescenteRESUMO
Nitrosomonas europaea is a chemolithotroph that obtains energy through the oxidation of ammonia to hydroxylamine while assimilates atmospheric CO2 to cover the cell carbon demands for growth. This microorganism plays a relevant role in the nitrogen biogeochemical cycle on Earth but its carbon metabolism remains poorly characterized. Based on sequence homology, we identified two genes (cbbG and gabD) coding for redox enzymes in N. europaea. Cloning and expression of the genes in Escherichia coli, allowed the production of recombinant enzymes purified to determine their biochemical properties. The protein CbbG is a glyceraldehyde-3-phosphate (Ga3P) dehydrogenase (Ga3PDHase) catalyzing the reversible oxidation of Ga3P to 1,3-bis-phospho-glycerate (1,3bisPGA), using specifically NAD+/NADH as cofactor. CbbG showed â¼6-fold higher Km value for 1,3bisPGA but â¼5-fold higher kcat for the oxidation of Ga3P. The protein GabD irreversibly oxidizes Ga3P to 3Pglycerate using NAD+ or NADP+, thus resembling a non-phosphorylating Ga3PDHase. However, the enzyme showed â¼6-fold higher Km value and three orders of magnitude higher catalytic efficiency with succinate semialdehyde (SSA) and NADP+. Indeed, the GabD protein identity corresponds to an SSA dehydrogenase (SSADHase). CbbG seems to be the only Ga3PDHase present in N. europaea; which would be involved in reducing triose-P during autotrophic carbon fixation. Otherwise, in cells grown under conditions deprived of ammonia and oxygen, the enzyme could catalyze the glycolytic step of Ga3P oxidation producing NADH. As an SSADHase, GabD would physiologically act producing succinate and preferentially NADPH over NADH; thus being part of an alternative pathway of the tricarboxylic acid cycle converting α-ketoglutarate to succinate. The properties determined for these enzymes contribute to better identify metabolic steps in CO2 assimilation, glycolysis and the tricarboxylic acid cycle in N. europaea. Results are discussed in the framework of metabolic pathways that launch biosynthetic intermediates relevant in the microorganism to develop and fulfill its role in nature.
Assuntos
Proteínas de Bactérias , Carbono/metabolismo , Gliceraldeído 3-Fosfato/metabolismo , Nitrosomonas europaea , Oxirredutases , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Dióxido de Carbono/metabolismo , Ciclo do Ácido Cítrico/fisiologia , Glicólise/fisiologia , Nitrosomonas europaea/enzimologia , Nitrosomonas europaea/genética , Oxirredução , Oxirredutases/genética , Oxirredutases/metabolismoRESUMO
Escherichia coli is still the preferred organism for large-scale production of recombinant proteins. The use of fusion proteins has helped considerably in enhancing the solubility of heterologous proteins and their purification with affinity chromatography. Here, the use of a small metal-binding protein (SmbP) from Nitrosomonas europaea is described as a new fusion protein for protein expression and purification in E. coli. Fluorescent proteins tagged at the N-terminal with SmbP showed high levels of solubility, compared with those of maltose-binding protein and glutathione S-transferase, and low formation of inclusion bodies. Using commercially available IMAC resins charged with Ni(II), highly pure recombinant proteins were obtained after just one chromatography step. Proteins may be purified from the periplasm of E. coli if SmbP contains the signal sequence at the N-terminal. After removal of the SmbP tag from the protein of interest, high-yields are obtained since SmbP is a protein of just 9.9 kDa. The results here obtained suggest that SmbP is a good alternative as a fusion protein/affinity tag for the production of soluble recombinant proteins in E. coli.