Mammalian membrane receptors expression as inclusion bodies in Escherichia coli.
Integral membrane proteins, in particular receptors, regulate numerous physiological functions. The primary difficulty presented by their study in vitro is to obtain them in sufficient amounts in a functional state. Escherichia coli is a host of choice for producing recombinant proteins for structural studies. However, insertion of G-protein coupled receptors into its plasma membrane usually results in bacterial death. An alternative approach consists of targeting recombinant receptors to inclusion bodies, where they accumulate without affecting bacterial growth, and then fold them in vitro . We describe here a general approach that consists of accumulating the receptor in bacterial inclusion bodies, then purifying it under denaturing conditions. A simple assay is then described to screen for refolding conditions of the protein.
Mouillac
Bernard
B
Institut de Génomique Fonctionnelle, Montpellier, France.
Banères
Jean-Louis
JL
D000818:Animals; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism*; D008322:Mammals / Q000235:genetics / Q000378:metabolism; D017510:Protein Folding; D043562:Receptors, G-Protein-Coupled / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D013917:Thrombin / Q000378:metabolism
D043562:Receptors, G-Protein-Coupled; D011994:Recombinant Proteins; D013917:Thrombin