RESUMEN
BACKGROUND: Second-generation ethanol production is a clean bioenergy source with potential to mitigate fossil fuel emissions. The engineering of Saccharomyces cerevisiae for xylose utilization is an essential step towards the production of this biofuel. Though xylose isomerase (XI) is the key enzyme for xylose conversion, almost half of the XI genes are not functional when expressed in S. cerevisiae. To date, protein misfolding is the most plausible hypothesis to explain this phenomenon. RESULTS: This study demonstrated that XI from the bacterium Propionibacterium acidipropionici becomes functional in S. cerevisiae when co-expressed with GroEL-GroES chaperonin complex from Escherichia coli. The developed strain BTY34, harboring the chaperonin complex, is able to efficiently convert xylose to ethanol with a yield of 0.44 g ethanol/g xylose. Furthermore, the BTY34 strain presents a xylose consumption rate similar to those observed for strains carrying the widely used XI from the fungus Orpinomyces sp. In addition, the tetrameric XI structure from P. acidipropionici showed an elevated number of hydrophobic amino acid residues on the surface of protein when compared to XI commonly expressed in S. cerevisiae. CONCLUSIONS: Based on our results, we elaborate an extensive discussion concerning the uncertainties that surround heterologous expression of xylose isomerases in S. cerevisiae. Probably, a correct folding promoted by GroEL-GroES could solve some issues regarding a limited or absent XI activity in S. cerevisiae. The strains developed in this work have promising industrial characteristics, and the designed strategy could be an interesting approach to overcome the non-functionality of bacterial protein expression in yeasts.
Asunto(s)
Isomerasas Aldosa-Cetosa/metabolismo , Chaperonina 60/genética , Proteínas de Escherichia coli/genética , Proteínas de Choque Térmico/genética , Ingeniería de Proteínas/métodos , Saccharomyces cerevisiae/genética , Isomerasas Aldosa-Cetosa/química , Isomerasas Aldosa-Cetosa/genética , Chaperonina 60/metabolismo , Proteínas de Escherichia coli/metabolismo , Etanol/metabolismo , Proteínas de Choque Térmico/metabolismo , Modelos Moleculares , Propionibacterium/enzimología , Conformación Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Xilosa/metabolismoRESUMEN
A tuberculose (TB) continua sendo um dos mais urgentes problemas de saúde pública do mundo, com 8 a 10 milhões de novos casos e 2 a 3 milhões de mortes a cada ano. Cerca de 50 milhõesde pessoas estão infectadas com o Mycobacterium tuberculosis. A fim de desenvolver testes para imunodiagnóstico da TB, vários antígenos têm sido testados na resposta imune humoral de pacientes com TB ativa. O teste imunoenzimático de ELISA realizado com amostras de plasma de 45 pacientes com tuberculose pulmonar e 172 contatos (96 prova tuberculínica negativa e 76, positiva) foi conduzido para avaliação da resposta imune humoral, com pesquisa de anticorpos das classesIgM e IgG contra o antígeno recombinante GroEs do Mycobacterium tuberculosis (MtGroEs). Pacientes com tuberculose pulmonar ativa apresentaram maiores níveis de IgG anti-rGroEs do que indivíduos saudáveis, o que permitiu a discriminação entre os dois grupos e sugeriu resposta imune humoral específica a este antígeno.
Tuberculosis (TB) remains one of the most important public health problems ofthe world, with 8-10 million new cases and 2-3 million deaths each year. About 50 million people are infected with Mycobacterium tuberculosis. In order to develop immunodiagnostic tests for TB, several antigens have been tested for the humoral immune response of patients with active TB. ELISA was performed with plasma samples from 45 patients with pulmonary TB and 172 contacts (96 negative and 76 positive for the tuberculin skin test). The humoral immune response was evaluated through assessment of IgM and IgG antibodies against recombinant Mycobacterium tuberculosis GroES (MtGroEs). Patients with active pulmonary tuberculosis had higher levels of IgG anti-rGroEs than healthy individuals, allowing discriminationbetween the two groups.
Asunto(s)
Humanos , Masculino , Femenino , Mycobacterium tuberculosis , Proteínas Recombinantes , Sistema Inmunológico , Tuberculosis Pulmonar , Ensayo de Inmunoadsorción Enzimática , Estudios TransversalesRESUMEN
Iron is an essential metal for all living organisms. However, iron homeostasis needs to be tightly controlled since iron can mediate the production of reactive oxygen species, which can damage cell components and compromise the integrity and/or cause DNA mutations, ultimately leading to cancer. In eukaryotes, iron-regulatory protein 1 (IRP1) plays a central role in the control of intracellular iron homeostasis. This occurs by interaction of IRP1 with iron-responsive element regions at 5' of ferritin mRNA and 3' of transferrin mRNA which, respectively, represses translation and increases mRNA stability. We have expressed IRP1 using the plasmid pT7-His-hIRP1, which codifies for human IRP1 attached to an NH2-terminal 6-His tag. IRP1 was expressed in Escherichia coli using the strategy of co-expressing chaperonins GroES and GroEL, in order to circumvent inclusion body formation and increase the yield of soluble protein. The protein co-expressed with these chaperonins was obtained mostly in the soluble form, which greatly increased the efficiency of protein purification. Metal affinity and FPLC ion exchange chromatography were used in order to obtain highly purified IRP1. Purified protein was biologically active, as assessed by electrophoretic mobility shift assay, and could be converted to the cytoplasmic aconitase form. These results corroborate previous studies, which suggest the use of folding catalysts as a powerful strategy to increase protein solubility when expressing heterologous proteins in E. coli.