RESUMO
G protein-coupled receptors (GPCRs) are notoriously difficult to express, particularly in microbial systems. Using GPCR fusions with the green fluorescent protein (GFP), we conducted studies to identify bacterial host effector genes that result in a general and significant enhancement in the amount of membrane-integrated human GPCRs that can be produced in Escherichia coli. We show that coexpression of the membrane-bound AAA+ protease FtsH greatly enhances the expression yield of four different class I GPCRs, irrespective of the presence of GFP. Using this new expression system, we produced 0.5 and 2 mg/L of detergent-solubilized and purified full-length central cannabinoid receptor (CB1) and bradykinin receptor 2 (BR2) in shake flask cultures, respectively, two proteins that had previously eluded expression in microbial systems.
Assuntos
Detergentes/química , Escherichia coli/metabolismo , Engenharia de Proteínas , Receptores Acoplados a Proteínas G/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Proteases Dependentes de ATP/biossíntese , Proteases Dependentes de ATP/genética , Membrana Celular/química , Membrana Celular/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/genética , Proteínas de Fluorescência Verde/biossíntese , Humanos , Receptor B2 da Bradicinina/biossíntese , Receptor B2 da Bradicinina/química , Receptor B2 da Bradicinina/isolamento & purificação , Receptor CB1 de Canabinoide/biossíntese , Receptor CB1 de Canabinoide/química , Receptor CB1 de Canabinoide/isolamento & purificação , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/isolamento & purificação , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , SolubilidadeRESUMO
A family of nanoparticles featuring surfaces of varying hydrophobicity was synthesized. The efficiency of DNA-binding was determined, demonstrating in a fivefold modulation in binding a 37-mer DNA strand. Nanoparticle-binding causes a reversible conformational change in the DNA structure, as demonstrated by circular dichroism and fluorescence experiments. Furthermore, the affinity of the nanoparticle for the DNA can be regulated by external agents, though stability of the complex is observed at relatively high ionic strengths.
Assuntos
DNA/metabolismo , Ouro/química , Nanoestruturas/química , Dicroísmo Circular , DNA/química , Ouro/metabolismo , Modelos Biológicos , Conformação de Ácido Nucleico , Oligonucleotídeos/química , Espectrometria de Fluorescência , Relação Estrutura-Atividade , Propriedades de SuperfícieRESUMO
Positively charged trimethylammonium-functionalized mixed monolayer protected clusters (MMPCs) bind DNA through complementary electrostatic interactions, resulting in complete inhibition of DNA transcription of T7 RNA polymerase. DNA was released from the nanoparticle by intracellular concentrations of glutathione, resulting in efficient transcription. The restoration of RNA production was dose-dependent in terms of GSH, with considerable control of the release process possible through variation in monolayer structure. This work presents a new approach to controlled release of DNA, with potential applications in the creation of transfection vectors and gene regulation systems.
