Process for producing a membrane protein
A process for the production of a membrane protein is provided. The process including the steps of: expressing a membrane protein in a host organism present in an aqueous medium, liberating the membrane protein from the host organism, adding a detergent solution to solubilize the membrane protein, recovering a liquid fraction of the solubilized membrane protein, subjecting the liquid fraction to chromatography to bind or retain the membrane protein on a stationary phase, and eluting the stationary phase with an elution buffer to produce the membrane protein. The process can produce relatively large amounts of membrane proteins in an efficient way without compromising the quality of the end product.
1 . A pilot or industrial scale process for producing a membrane protein comprising the steps of:
a. expressing said membrane protein in a host organism present in an aqueous medium,
b. i) liberating the membrane protein from the host organism by mechanically lysing cells of the host organism to form lysed cell membrane fragments that include the membrane protein and ii) isolating the lysed cell membrane fragments including the membrane protein in the form of a pellet by a first centrifugation,
c. adding a detergent solution to a resuspension of the pellet obtained in b to solubilize the membrane protein to form a solubilized membrane protein,
d. recovering a liquid fraction of the solubilized membrane protein obtained in c as a supernatant by a second centrifugation,
e. subjecting the liquid fraction to chromatography to bind or retain the solubilized membrane protein on a stationary phase, and
f. eluting the protein from the stationary phase to produce the solubilized membrane protein, wherein the first and second centrifugations in steps b and d are each performed at 500 g to 30,000 g and the process does not include ultracentrifugation of the liquid fraction obtained in steps b and d.
2 . The process according to claim 1 , wherein the aqueous medium comprising the host organism of step a is filtered prior to the liberating of the membrane protein from the host organism according to step b through a microfiltration membrane having a pore diameter of 0.5 micrometer or less.
3 . The process according to claim 2 , wherein the host organism is isolated after the aqueous medium comprising the host organism is filtered through the microfiltration membrane and by a third centrifugation of the aqueous medium comprising the host organism to form an isolated host organism.
4 . The process according to claim 3 , wherein the isolated host organism is washed with an isotonic saline solution to dissolve contaminating salts and by a fourth centrifugation of the isotonic saline solution including the isolated host organism to isolate a washed host organism.
5 . The process according to claim 1 , wherein a dilution buffer is added prior to step b.
6 . The process according to claim 1 , wherein a cationic flocculant is added to the solubilized membrane protein that is obtained in c to form a suspension.
7 . The process according to claim 6 , wherein the cationic flocculant is a polyamine compound.
8 . The process according to claim 6 , wherein the cationic flocculant is allowed to react with cell parts of the suspension during agitation to form flocs, wherein the liquid fraction of step d is recovered as the supernatant from an additional centrifugation of the suspension containing the flocs.
9 . The process according to claim 1 , wherein the detergent solution added in c is selected from the group consisting of lauryl dimethylamine N-oxide (LDAO), octyl glucoside (OG), dodecyl maltoside (DDM) and combinations thereof.
10 . The process according to claim 1 , wherein the second centrifugation of step d is performed at 1,000 g to 10,000 g.
11 . The process according to claim 1 , wherein the membrane protein is associated with a first part of an affinity pair and the stationary phase is associated with a second part of the affinity pair.
12 . The process according to claim 11 , wherein the first part of the affinity pair is a histidine tag comprising 8 or more histidine molecules.
13 . The process according to claim 1 , wherein step f includes eluting with an elution buffer which comprises imidazole.
14 . The process according to claim 13 , wherein the stationary phase capable of binding or retaining the solubilized membrane protein is present in a column and the column prior to elution with the elution buffer, is washed with a washing buffer comprising 40% or less of a concentration of imidazole in the elution buffer.
15 . The process according to claim 13 , wherein a concentration of imidazole in the elution buffer is 400 mM or more.
16 . The process according to claim 1 , wherein a cationic flocculant is added to the lysed cell membrane fragments that are obtained in b i) to form a suspension.
17 . The process according to claim 6 , wherein the cationic flocculant is allowed to react with the lysed cell membrane fragments of the suspension during agitation to form flocs of the lysed cell membrane fragments including the membrane protein and the pellet in b ii) is formed by separating the flocs from the suspension by the first centrifugation.