Purification of proteins
OwnerMarcus Wolf William HARTMANN, Ingo ALDAG +3inventors
InventorsMarcus Wolf William HARTMANN, Ingo ALDAG, Jan ROSSDORF, Stefan UTHOFF, Florian SCHUMACHER
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Abstract
The present invention relates to a method for purifying proteins, which proteins are produced by homologous or heterologous expression in a ciliate host, from harvested cell culture fluid (HCCF) which was not chromatographically purified. The method comprises the steps of incubating a harvested cell culture fluid with a kosmotropic agent, efflorescing protein by formation of crystals, and, optionally, harvesting effloresced protein.
Claims31
A method for purifying proteins, which proteins are produced by homologous or heterologous expression in a ciliate host, from harvested cell culture fluid (HCCF) which was not chromatographically purified,
- the method comprising the steps of
- a) incubating a harvested cell culture fluid with a kosmotropic agent,
- b) efflorescing protein by formation of crystals, and
- c) optionally, harvesting effloresced protein,
- wherein the method does not comprise a step of crosslinking the proteins before or after efflorescing.
The method according to any one of the aforementioned claims, wherein the harvested cell culture fluid comprises between ≥3 and ≤200 g/l of protein.
The method according to any one of the aforementioned claims, wherein for efflorescing the protein,
- a pH of between ≥5 and ≤8 is established in the medium, and/or
- a temperature of between ≥10 and ≤40° C. is established.
The method according to any one of the aforementioned claims, wherein, for the incubation of the harvested cell culture fluid, a kosmotropic salt concentration is established in the range of between ≥50 and ≤2500 mM.
The method according to any one of the aforementioned claims, wherein, for the incubation of the harvested cell culture fluid, a polyalcohol concentration is established in the range of between ≥2 and ≤25% w/v.
The method according to any one of the aforementioned claims, wherein the incubation of the harvested cell culture fluid with the kosmotropic agent is carried out over a period of between ≥10 min and ≤36 hrs.
The method according to any one of the aforementioned claims, wherein a pre-harvest cell culture fluid is subjected to one or more filtration and/or centrifugation steps prior to step a), to obtain the harvested cell culture fluid.
The method according to any one of the aforementioned claims, wherein the ciliate host is Tetrahymena sp.
The method according to any one of the aforementioned claims, wherein the homologous or heterologous expression is under control of
- a metallothionein gene (MTT1) inducible promoter, and/or
- a heat inducible promoter.
The method according to any one of the aforementioned claims, wherein the protein is an enzyme.
The method according to any one of the aforementioned claims, wherein the enzyme is
- a) Tetrahymena lipase according to any one of SEQ ID NOs 1-3 or 8-10,
- b) Tetrahymena amylase according to SEQ ID NOs 4 or 11,
- c) Tetrahymena protease according to any of SEQ ID NOs 5-7, 12-14 or 15-17, or
- d) a variant of any one of the above enzymes having
- (i) at least 90% amino acid sequence identity therewith, wherein said variant retains enzyme functionality, and/or
- (ii) between 1 and 3 amino acid residues removed or added at the N-terminus and/or the C terminus
A purified, crystallized protein or pharmaceutical intermediate which comprises a ciliate-type glycosylation pattern.
Dependencies are inferred from the claim text.