RESUMO
Immobilized metal affinity chromatography has recently been used for purification of histidine-tagged membrane proteins in the presence of detergents with varying success. Strong binding to the metal resin is essential for purification when expression levels are low. We have investigated the influence of tag length and type of detergent on the purification of a neurotensin receptor fusion protein expressed in Escherichia coli at a level of about 0.1% of membrane protein. Receptors with six C-terminal histidine residues did not bind to nickel resin in the presence of the anionic detergent sodium dodecyl sulfate. In contrast, partial purification assessed by densitometry of Coomassie-stained gels was achieved using the nonionic detergents dodecyl maltoside or Triton X-100 (53% pure), or a detergent mixture containing the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (46% pure). Linking a highly charged epitope tag to the histidine tail did not affect the nickel-binding properties of receptors. The level of purification was substantially improved (72% pure) by extending the histidine tail to 10 residues because this allowed stringent washes at high imidazole concentration to remove nonspecifically bound contaminants. This strategy not only resulted in efficient purification of receptors from crude membranes, but also worked particularly well for single-step purification from total cell lysates, resulting in 340-fold purification of functional neurotensin receptor.
Assuntos
Cromatografia de Afinidade/métodos , Receptores de Neurotensina/isolamento & purificação , Detergentes , Epitopos , Escherichia coli , Níquel , Oligopeptídeos , Peptídeos , Receptores de Neurotensina/genética , Proteínas Recombinantes de Fusão/isolamento & purificaçãoRESUMO
Bacteriophage PRD1 is a membrane-containing phage which could be used for expression of foreign membrane proteins such as neurotensin receptor (NTR), a seven-helix G-protein coupled receptor. To ensure recognition of NTR by the phage system six different fusion genes were constructed, each encoding a different phage integral membrane protein fused to the N-terminus of NTR, and expression of the fusion proteins in Escherichia coli was analysed. Here we report the identification of two fusion constructs that retained the function of NTR in E. coli. This provides the basis to develop the phage system as a heterologous expression system for seven-helix receptors.
Assuntos
Bacteriófago P1/metabolismo , Escherichia coli/genética , Proteínas de Membrana/metabolismo , Receptores de Neurotensina/genética , Sequência de Aminoácidos , Animais , Bacteriófago P1/genética , Sítios de Ligação , Western Blotting , Eletroforese em Gel de Poliacrilamida , Proteínas de Ligação ao GTP/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Genes myc/genética , Genoma Bacteriano , Proteínas de Membrana/química , Dados de Sequência Molecular , Ratos , Receptores de Neurotensina/biossíntese , Receptores de Neurotensina/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
With the goal of obtaining sufficient quantities of seven-helix G-protein-coupled receptors for structural analysis, we have studied the functional expression of a rat neurotensin receptor cDNA in Escherichia coli with and without a signal sequence and as a fusion with the gene coding for maltose-binding protein. The addition of an N-terminal signal peptide resulted in increased expression levels. In vitro translation at a high level revealed that the codon usage of the rat neurotensin receptor cDNA was not critical for overproduction. Expression of neurotensin receptor cDNA fused to the 3' end of the gene encoding maltose-binding protein resulted in a 40-fold increase in neurotensin-binding sites. Binding of [3H]neurotensin to intact bacteria or E. coli membranes was saturable, with a dissociation constant, KD, of 0.23 nM (Bmax. = 450 sites/bacterium or 15 pmol/mg of crude membrane protein). The binding properties of all recombinant receptors presented in this study were similar and corresponded to those of the high-affinity binding sites in rat brain. For immunological detection and future purification of neurotensin receptor, a C-terminal pentahistidine/c-myc tail was introduced. Western-blot analysis revealed the association of neurotensin receptor with E. coli membranes.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Proteínas de Escherichia coli , Proteínas de Transporte de Monossacarídeos , Receptores de Neurotensina/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Clonagem Molecular , Cisteína/genética , DNA Complementar , Escherichia coli , Leucina/genética , Maltose/metabolismo , Proteínas Ligantes de Maltose , Dados de Sequência Molecular , Biossíntese de Proteínas , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The gene encoding the Rhodopseudomonas viridis cytochrome c2 (cycA) has been introduced on a broad host range vector into Paracoccus denitrificans, leading to high-level expression of the holo-cytochrome with the heme moiety covalently attached to the apoprotein. The cytochrome was demonstrated to reside in the periplasmic space of the host cell. In contrast to R. viridis, aerobic rather than anaerobic growth conditions led to higher production levels of the holo-cytochrome in P. denitrificans. This heterologous expression system provides a suitable genetic background for the functional expression and mutagenesis of polypeptides involved in bacterial photosynthesis, offering the possibility of detailed structural and functional investigation.