PROCESS FOR PRODUCING A MEMBRANE PROTEIN

Abstract

A process for the production of a membrane protein 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 including the quality of the end product.

Claims

1. A process for producing a membrane protein comprising the steps of: a. Expressing a membrane protein in a host organism present in an aqueous medium, b. Liberating the membrane protein from the host organism, c. Adding a detergent solution to solubilize the membrane protein, d. Recovering a liquid fraction of the solubilized membrane protein as the supernatant by centrifugation, e. Subjecting the liquid fraction to chromatography to bind or retain the membrane protein on a stationary phase, and f. Eluting the stationary phase with an elution buffer to produce the membrane protein, wherein the centrifugation in step d is performed at 500 g to 30,000 g.

2-26. (canceled)

27. 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 host organism according to step b through a microfiltration membrane having a pore diameter of 0.5 micrometer or less.

28. The process according to claim 27, wherein the host organism is isolated, optionally after the filtration, by centrifugation of the aqueous medium comprising the host organism, said centrifugation being performed at 500 g to 30.000 g.

29. The process according to claim 28, wherein the isolated host organism is washed with an isotonic saline solution to dissolve contaminating salts and subsequently centrifuged to isolate the washed host organism.

30. The process according to claim 1, wherein a dilution buffer is added prior to step b.

31. The process according to claim 1, wherein an aqueous lysis solution is added to liberate the membrane protein from the host organism.

32. The process according to claim 31, wherein the aqueous lysis buffer is a detergent solution, which simultaneously solubilizes the membrane protein.

33. The process according to claim 1, wherein the liberation of the membrane protein from the host cells is performed by a homogenizer.

34. The process according to claim 1, wherein a cationic flocculant is added to the liberated membrane protein to form a suspension.

35. The process according to claim 34, wherein the cationic flocculant is a polyamine compound.

36. The process according to claim 34, wherein the cationic flocculant is allowed to react with the cell parts of the resuspension during gentle agitation to form flocs, wherein the liquid fraction of step d is recovered as the supernatant from a centrifugation of the suspension containing the flocs and.

37. The process according to claim 1, wherein the liquid fraction of step d is recovered from a resuspension of a solid fraction, wherein the solid fraction is resuspended in a detergent solution for solubilizing the membrane protein.

38. The process according to claim 37, wherein the liquid fraction of step d is harvested as the supernatant by centrifugation.

39. The process according to claim 37, wherein the detergent is selected from the group consisting of lauryl dimethylamine N-oxide (LDAO), octyl glucoside (OG), dodecyl maltoside (DDM) or combinations thereof.

40. The process according to claim 1, wherein the centrifugation of step d is performed at 1,000 g to 10,000 g.

41. 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.

42. The process according to claim 41, wherein the first part of the affinity pair is a histidine tag comprising 8 or more histidine molecules.

43. The process according to claim 1, wherein the eluting buffer comprises imidazole.

44. The process according to claim 43, wherein the stationary phase capable of binding or retaining the membrane protein is present in a column and the column prior to the elution with the elution buffer, is washed with a washing buffer comprising 40% or less of the concentration of imidazole in the elution buffer.

45. (canceled)

46. The process according to claim 43, wherein the concentration of imidazole in the elution buffer is 400 mM or more.

Description

EXAMPLES

Example 1

[0046] An E. coli BL21 strain is prepared comprising a vector producing aquaporin proteins linked to a His tag at the C-terminal. The His tag contains ten consecutive histidine molecules attached to the primary sequence of the aquaporin membrane protein.

[0047] The E. coli strain is nurtured in a standard medium to obtain a total fermentation broth of 150 L. The E. coli cells was harvested by filtering the fermentation broth through a microfiltration membrane having a pore diameter of 0.05 μm. The filtrate containing the E. coli cells is reduced to about 50 L and subsequently subjected to centrifugation at 5300 g for 20 minutes. Thus, the E. coli cells are up-concentrated by microfiltration and the remaining medium is subsequently removed as the supernatant by centrifugation.

[0048] The pellets obtained by the centrifugation are collected and added 1:1 volume 0.9% sodium chloride to wash the cells and dissolve contaminating salts. Subsequently, the washing solution is removed in a centrifuge running at 5300 g for 20 minutes. The supernatant was discarded and the washed cells collected as pellets. The pellets may be stored by freezing at −20° C. or used directly in the next step.

[0049] The pellets comprising the E. coli cells were solubilized in about 47 L TRIS buffer used as a binding buffer. After stirring for about one hour 6.4 L detergent (5% LDAO) was added for solubilization to a final concentration of 0.6%. The mixture was incubated overnight at room temperature with gentle stirring. By the resuspension of the cells in a buffer containing a detergent a cell lysis occurred. By the cell lysis membrane proteins are liberated from the internal membranes of the cells and solubilized by the detergent.

[0050] To remove negatively charged cell material a polyamine (Superfloc C581) was added in a volume of 427 mL. The mixture was incubated for 30 minutes with stirring at room temperature for coagulating the negatively charged molecules such as cell debris, DNA, RNA and to a certain extent of proteins. The membrane protein solubilized by the detergent remains in the aqueous phase. The mixture is centrifuged at a maximum speed of 5300 g for 15 minutes. The pellet containing the cell debris, DNA, RNA and solubilized proteins are discarded and the supernatant collected. The supernatant is transferred to a container containing 107 L dilution buffer to obtain a final volume of 160 L with a final concentration of 0.2% LDAO.

[0051] A column containing the affinity resin “Capto Chelating” from GE healthcare is provided. The resin is charged with Ni2+ which binds to the His10 tag. The column was loaded with the diluted supernatant and subsequently washed with 10 column volumes wash buffer containing 200 mM imidazole to wash out the nonspecific binding components. Subsequently, 2.5 column volumes elution buffer of aqueous 1000 mM imidazole was used to release the membrane protein from the column. The protein eluted from the column was tested by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), which primarily showed a single band indicating a purity of above 80%.

Example 2

[0052] An E. coli strain BL21 is prepared comprising a vector producing aquaporin proteins linked to a His tag at the C-terminal. The His tag contains ten consecutive histidine molecules attached to the primary sequence of the aquaporin membrane protein.

[0053] The E. coli strain is nurtured in a standard medium to obtain a total fermentation broth of 250 L. The E. coli cells were harvested by filtering the fermentation broth through a PES flat sheet membrane having a pore diameter of 0.05 μm. The filtrate containing the E. coli cells is reduced to about 50 L and subsequently subjected to centrifugation at 5300 g in a Sorvall 16 L centrifuge for 20 minutes. Thus, the E. coli cells are up-concentrated by microfiltration and the remaining medium is subsequently removed as the supernatant by centrifugation. The pellets may be stored by freezing at −20° C. or used directly in the next step.

[0054] The pellets comprising the E. coli cells were resuspended in about 50 L buffer (aqueous solution of the protease inhibitor PMSF and EDTA) and homogenized at 1000 bar in a Stansted nm-GEN 7575 homogenizer. To isolate the cell material of interest a polyamine (Superfloc C581) is added in a concentration of 12 ml/L. The temperature was maintained around 10-15° C. The mixture was incubated for 30 minutes with stirring at room temperature. The mixture was centrifuged at a maximum speed of 5300 g for 30 minutes. The pellet contains the membrane protein and the supernatant is discarded.

[0055] The pellet was resuspended in a 0.9% sodium chloride solution to obtain a total protein concentration of approximately 50 mg/ml. Solubilization of the membrane protein was performed by adding 28 L TRIS binding buffer and 4.5 liter 5% LDAO to 5 L of the resuspended pellet material. At room temperature and gentle stirring the mixture was allowed to incubate for 2 to 24 hours.

[0056] After the solubilization process the mixture was centrifuged in 2 L containers at 5300 g for 90 minutes. The supernatant was recovered and the LDAO concentration was adjusted to 0.2% by addition of dilution buffer.

[0057] A column containing the affinity resin “Capto Chelating” from GE healthcare is provided. The resin is charged with Ni.sup.2+ which binds to the His10 tag. The column was equilibrated by loading binding buffer having 0.2% LDAO. Subsequently, the column was loaded with the diluted supernatant and washed with 10 column volumes wash buffer containing 200 mM imidazole to wash out the nonspecific binding components. Subsequently, 2.5 column volumes elution buffer of aqueous 1000 mM imidazole is used to release the membrane protein from the column. The protein eluted from the column was tested by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) chromatography, which showed only a single band indicating a purity of above 95%.

Example 3

[0058] An E. coli strain BL21 is prepared comprising a vector producing aquaporin proteins linked to a His tag at the C-terminal. The His tag contains ten consecutive histidine molecules attached to the primary sequence of the aquaporin membrane protein.

[0059] The E. coli strain is nurtured in a standard medium to obtain a total fermentation broth of 250 L. The fermentation batch had an OD600 13 when harvested, and it was induced for 42.5 hours. The material was homogenized twice at 100 MPa in a Stansted nm-GEN 7575 homogenizer. 10 mL was taken from the lysed material and added to a 15 mL falcon tube.

[0060] Centrifugation was then carried out for 1 hour with 5300 g and the pellet was separated from the supernatant. The pellet was then resuspended in 10 mL 0.9% NaCl. BCA assay was used to determine the concentration (mg/mL total protein) of the resuspended pellets and the separated supernatants after the first spin.

[0061] The resuspended pellets and the separated supernatants were finally solubilized (1 mL) for 2 hours in 0.6% LDAO (120 uL of 5% LDAO stock from Carbosynth was used to 1 mL material) and were centrifuged again at 5300 G for 15 minutes. Once again, the supernatants were separated from the pellets. The pellets were resuspended (up to 1 mL again) in binding buffer w/o LDAO.

[0062] All samples were analyzed on SDS-gel, which showed that around 60% of the proteins in the supernatant was the aquaporin membrane protein.

Example 4

[0063] Following the purification of the Aquaporin Z in example 2, the size distribution of the solubilized protein in LDAO was measured.

[0064] A comparison was made between the same elution buffer, with and without protein, to evaluate whether the particle size distribution was influenced by the buffer components (including detergent) or the membrane protein.

[0065] The Aquaporin Z protein was eluted from the IMAC column using Elution buffer with 1000 mM imidazole and 0.2% w/v LDAO. The purified protein was measured for its protein concentration by amino acid analysis. 6.44 mg/mL of clear, solubilized, protein in Elution buffer was loaded into 3 separate cuvettes (Sarstedt, 4 mL, PMMA, ca. no. 67.755, Numbrecht, Germany) and analyzed for the size distribution by the Malvern Zetasizer Nano-ZS (Malvern Instruments Ltd., Malvern UK) using the Malvern Zetasizer software v.7.02. Results presented in table 1 are the average of the samples run in triplicate.

TABLE-US-00001 TABLE 1 Concentration Size, diameter (d .Math. nm) of AqpZ Population intensity (%) Sample (mg/mL) Pop. 1 Pop. 2 Pop. 3 Purified AqpZ 6.44 315.6 ± 108 ± 17.1 ± in Elution 116.8 nm 32.3 nm 2.5 nm buffer (avg.) 45.9% 35.5% 14.9% Elution buffer 0 0.85 ± 8.6 ± 103 ± (avg.) 0.16 nm 1.2 nm 5.5 nm 56.8% 41.1%  2.1%

[0066] The Elution buffer sample (no protein) contains a population distribution with 97.9% of the particles with a size of 8.6 nm or smaller, indicating there are no large detergent micelles in solution that would be retained by microfiltration.

[0067] 81.4% of the particles in the sample with protein show a size of 108 nm or larger, thereby indicating that the presence of the protein and detergent together form large soluble particles that are retained during microfiltration. The experiment surprisingly shows that large, stable, particles composed of LDAO micelles and membrane protein are produced.

Example 5

Expression of Histidine Tagged Aquaporin From Oryza sativa Japonica (Japanese Rice) in Escherichia coli and its Purification Using IMAC

[0068] The gene encoding aquaporin from Oryza sativa Japonica (UNIPROT: A3C132) was codon optimized using Geneart's (Subsidiary of Thermo Fischer Scientific) service for improving expression in E. coli. The resulting gene was synthesized with the addition of ten histidine encoding codons C-terminally, along with flanking Ndel/Xhol restriction sites N-terminally and C-terminally, respectively (Gene ID: aquaporin_Oryza_sativa_Japonica). The synthetic gene fragment was digested with Ndel/Xhol restriction enzymes and ligated to Ndel/Xhol—digested and purified vector pUP1909 fragment. The resulting ligation mixture was transformed into Escherichia coli DH10B and kanamycin resistant transformants were selected on LB agar plates with kanamycin. Transformants were confirmed by sequencing of the genetic construct. Isolated vector DNA was subsequently transferred to the production host, Escherichia coli BL21.

[0069] In order to heterologously express aquaporin in E. coli, the production host was grown in minimal medium consisting of 30 g/L Glycerol, 6 g/L (NH.sub.4).sub.2HPO.sub.4, 3 g/L KH.sub.2PO.sub.4, 5 g/L NaCl, 0.25 g/L MgSO.sub.4.7H.sub.2O, 0.4 g/L Fe(III)citrate and 1 mL/L sterile filtered trace metal solution. The trace metal solution consisted of 1 g/L EDTA, 0.8 g/L CoCl.sub.2.6H.sub.2O, 1.5 MnC.sub.2.4H.sub.2O, 0.4 g/L CuCl.sub.2.2H.sub.2O, 0.4 g/L H.sub.3BO.sub.3, 0.8 g/L Na.sub.2MoO.sub.4.2H.sub.2O, 1.3 g/L Zn(CH.sub.3OO)2.2H.sub.2O. After inoculation and overnight growth, additional 0.25 g/L MgSO.sub.4.7H.sub.2O was added.

[0070] E. coli was cultivated in 3L Applikon Bioreactors with ez-Control in a batch fermentation process. Protein production was induced by addition of IPTG to a final concentration of 0.5 mM at an optical density (OD 600 nm) of approximately 30. The culture was induced for approximately 24 hours and the bacterial cells were harvested with centrifugation at 5300 g for 20 min.

[0071] The pellets comprising the E. coli cells were resuspended in buffer (aqueous solution of the protease inhibitor PMSF and EDTA) and homogenized at 1000 bar in a Stansted nm-GEN 7575 homogenizer. The temperature was maintained around 10-15° C. The mixture was centrifuged at a maximum speed of 5300 g for 30 minutes. The pellet contains the membrane protein and the supernatant is discarded.

[0072] The pellet was resuspended in a 0.9% sodium chloride solution to obtain a total protein concentration of approximately 50 mg/ml. Solubilization of the membrane protein was performed by adding 28 L TRIS binding buffer and 4.5 liter 5% LDAO to 5 L of the resuspended pellet material. At room temperature and gentle stirring the mixture was allowed to incubate for 2 to 24 hours.

[0073] After the solubilization process the mixture was centrifuged in 2 L containers at 5300 g for 90 minutes. The supernatant was recovered and the LDAO concentration was adjusted to 0.2% by addition of dilution buffer.

[0074] After solubilization and clarification, the protein was captured using IMAC and eluted in Elution buffer containing 1000 mM imidazole and 0.2% w/v LDAO. The elution fractions were analyzed by SDS Page and only revealed a single major band which migrated at 27 kDa which corresponds to the size of aquaporin from Japanese rice. Furthermore, the result was confirmed by comparison to a negative control purification from E. coli transformed with an empty vector. The negative control resulted in no purified protein. Western blot analysis with antibodies (TaKaRa Bio) specific for the histidine-tag resulted as expected in a clear signal from the purified protein and no signal from the negative control confirming the origin of the purified protein as the histidine tagged membrane protein.

Example 6

Expression of Histidine Tagged Aquaporin From Eucalyptus grandis in Escherichia coli and its Purification Using IMAC

[0075] The gene encoding aquaporin from Eucalyptus grandis (UNIPROT: A0A059C9Z4) was codon optimized using Geneart's (Subsidiary of Thermo Fischer Scientific) service for improving expression in E. coli. The resulting gene was synthesized with the addition of ten histidine encoding codons C-terminally, along with flanking Ndel/Xhol restriction sites N-terminally and C-terminally, respectively (Gene ID: aquaporin_Eucalyptus_grandis). The gene was cloned and expressed as described in Example 5. The protein was successfully purified and confirmed as described in Example 5.

Example 7

Expression of Histidine Tagged Aquaporin From Solanum tuberosum (Danish Potato) in Escherichia coli and its Purification Using IMAC

[0076] The gene encoding aquaporin from Solanum tuberosum (UNIPROT: Q38HT6) was codon optimized using Geneart's (Subsidiary of Thermo Fischer Scientific) service for improving expression in E. coli. The resulting gene was synthesized with the addition of ten histidine encoding codons C-terminally, along with flanking Ndel/Xhol restriction sites N-terminally and C-terminally, respectively (Gene ID: aquaporin_Solanum_tuberosum). The gene was cloned and expressed as described in Example 5. The protein was successfully purified and confirmed as described in Example 5.

Example 8

Expression of Histidine Tagged Aquaporin From Milnesium tardigradum (Water Bear) in Escherichia coli and Purifying Using IMAC

[0077] The gene encoding aquaporin from Milnesium tardigradum (UNIPROT: G5CTG2) was codon optimized using Geneart's (Subsidiary of Thermo Fischer Scientific) service for improving expression in E. coli. The resulting gene was synthesized with the addition of ten histidine encoding codons C-terminally, along with flanking Ndel/Xhol restriction sites N-terminally and C-terminally, respectively (Gene ID: aquaporin_Milnesium_tardigradum). The gene was cloned and expressed as described in Example 5. The protein was successfully purified and confirmed as described in Example 5.

Example 9

Expression of Histidine Tagged Aquaporin From Halomonas sp. in Escherichia coli and its Purification Using IMAC

[0078] The gene encoding aquaporin from Halomonas sp. (UNIPROT: A0A2N0G6U6) was codon optimized using Geneart's (Subsidiary of Thermo Fischer Scientific) service for improving expression in E. coli. The resulting gene was synthesized with the addition of ten histidine encoding codons C-terminally, along with flanking Ndel/Xhol restriction sites N-terminally and C-terminally, respectively (Gene ID: aquaporin_Halomonas_sp). The gene was cloned and expressed as described in Example 5. The protein was successfully purified and confirmed as described in Example 5.

Example 10

Expression of Histidine Tagged Aquaporin From Kyrpidia spormannii in Escherichia coli and its Purification Using IMAC

[0079] The gene encoding aquaporin from Kyrpidia sp. (UNIPROT: A0A2K8N5Z5) was codon optimized using Geneart's (Subsidiary of Thermo Fischer Scientific) service for improving expression in E. coli. The resulting gene was synthesized with the addition of ten histidine encoding codons C-terminally, along with flanking Ndel/Xhol restriction sites N-terminally and C-terminally, respectively (Gene ID: aquaporin_Kyrpidia_spormannii). The gene was cloned and expressed as described in Example 5. The protein was successfully purified and confirmed as described in Example 5.

Example 11

Expression of Histidine Tagged Aquaporin From Methanothermobacter sp. in Escherichia coli and its Purification Using IMAC

[0080] The gene encoding aquaporin from Methanothermobacter sp. (UNIPROT: A0A223ZCQ2) was codon optimized using Geneart's (Subsidiary of Thermo Fischer Scientific) service for improving expression in E. coli. The resulting gene was synthesized with the addition of ten histidine encoding codons C-terminally, along with flanking Ndel/Xhol restriction sites N-terminally and C-terminally, respectively (Gene ID: aquaporin_Methanothermobacter_sp). The gene was cloned and expressed as described in Example 5. The protein was successfully purified and confirmed as described in Example 5.

Example 12

Expression of Histidine Tagged Aquaporin From Novibacillus thermophilus in Escherichia coli and its Purification Using IMAC

[0081] The gene encoding aquaporin from Novibacillus thermophilus (UNIPROT: A0A1 U9K5R2) was codon optimized using Geneart's (Subsidiary of Thermo Fischer Scientific) service for improving expression in E. coli. The resulting gene was synthesized with the addition of ten histidine encoding codons C-terminally, along with flanking Ndel/Xhol restriction sites N-terminally and C-terminally, respectively (Gene ID: aquaporin_Novibacillus_thermophilus). The gene was cloned and expressed as described in Example 5. The protein was successfully purified and confirmed as described in Example 5.

Example 13

Expression of Outer Membrane Protein A (OmpA) From Escherichia coli in E. coli

[0082] Expression and purification of OmpA (UNIPROT: P0A910) in E. coli was carried out essentially as described in Example 5, except that E. coli was transformed with an empty vector and solubilization was prolonged to 24 hours. The resulting solubilized protein was estimated to a purity of approximately 30-50% by SDS-PAGE, thus clearly stating that OmpA was successfully isolated in the membrane fraction following the standard purification procedure. An additional size exclusion chromatography (SEC) step could have further purified OmpA.

Example 14

Constructing a Saccharomyces cerevisiae Strain Expressing Aquaporin-Z (AqpZ) From Escherichia coli Fused to an N-Terminal Histidine-Tagged yEGFP and Separated by a Tobacco Etch Virus (TEV) Protease Cleavage Site

[0083] AqpZ (UNIPROT ID: P60844) from E. coli was codon optimized for expression in S. cerevisiae using Geneart's service for improved expression in S. cerevisiae. For expression in S. cerevisiae, AqpZ was fused with yeast enhanced green fluorescent protein (yEGFP) N-terminally (Brendan P. Cormack et al, Microbiology (1997), 143, 303-311) to enable visual detection and quantification of membrane protein expression. Furthermore, eight histidines (His8) was added N-terminally to yEGFP as an IMAC purification tag. The His8-yEGFP and AqpZ was genetically separated by a TEV protease cleavage site incorporated by PCR primers during construction.

[0084] Rapid and efficient construction of the plasmid encoding His8-yEGFP-TEV-AqpZ fusion was carried out by in vivo homologous recombination of overlapping regions incorporated by the primers in S. cerevisiae between a His8-yEGFP-TEV PCR fragment, a TEV-AqpZ PCR fragment, and the linearized expression plasmid derived from SalI, HindIII and BamHI digestion of the pEMBLyex4 plasmid, as described in (Scientific Reports 7: 16899). The TEV cleavage site enables subsequent removal of the His8-yEGFP protein by the TEV protease.

[0085] Selection of S. cerevisiae transformants was carried out on minimal medium plates deficient in uracil but supplemented with leucine and lysine to ensure survival of bacterial cells with correctly recombined DNA fragments. Medium composition and amino acid concentrations used in this example were identical to the ones listed in Example 15.

Example 15

Expression of His8-yEGFP-TEV-AqpZ with Saccharomyces cerevisiae.

[0086] A single colony of transformed yeast cells was selectively propagated until saturation in 5 ml of glucose minimal medium supplemented with 60 mg/L leucine and 30 mg/L lysine. 200 μl of this culture was subsequently propagated in 5 ml of glucose minimal medium supplemented with 30 mg/L lysine for selection for high plasmid copy numbers. Freeze stocks of the high plasmid copy number cells were prepared.

[0087] 200 μl of a thawed freeze-stock was added to 10 ml of minimal medium supplemented with lysine and grown until saturation. 1 ml of the culture was transferred to 100 ml of the same medium. After overnight growth, an aliquot corresponding to a final OD600 of 0.05 was transferred to 1.5 liters of minimal medium with an initial concentration of 20 g/L glucose as carbon source and 30 g/L glycerol supplemented with extra amino acids. The culture was grown in a 3 L Applikon® bioreactor equipped with ez-Control connected to a PC running Lucullus® software (Applikon, Holland, and SecureCell, Switzerland).

[0088] The initial part of the fermentation was performed at 20° C. in minimal medium. The bioreactor was fed with glucose to a final concentration of 3% w/v when the initial amount of glucose had been metabolized. The pH of the growth medium was kept at 6.0 by computer-controlled addition of 1 M NH.sub.4OH. When the CO.sub.2-exhaust gas leveled off, due to limited access of glucose, the bioreactor was cooled to 15° C. prior to induction of recombinant AQP production. Addition of 50 mL/L expression medium consisting of 400 mL/L ASD-10, 400 mL/L extra amino acids, 200 g/L glycerol and 20 g/L galactose started the expression of the recombinant protein. Yeast cells were harvested after ˜96 h.

[0089] The minimal medium consisted of 20 g/L glucose, 100 mL/L ASD-10, 5 mL/L V-200, 30 g/L glycerol, 0.1 g/L Ca.sub.2Cl. The ASD-10 consisted of 50 g/L (NH.sub.4).sub.2SO.sub.4, 8.75 g/L KH.sub.2PO.sub.4, 1.25 g/L K.sub.2HPO.sub.4, 5 g/L MgSO.sub.4.7H.sub.2O, 1 g/L NaCl, 5 mg/L H.sub.3BO.sub.3, 1 mg/L KI, 4 mg/L MnSO.sub.4.1H.sub.2O, 4.2 mg/L ZnSO.sub.4.7H.sub.2O, 0.4 mg/L CuSO.sub.4.5H.sub.2O, 2 mg/L FeCl.sub.3 and 2 mg/L Na.sub.2MoO.sub.4.2H.sub.2O. The V-200 consisted of 4 mg/L Biotin, 400 mg/L D-pantothenic acid, 0.4 mg/L Folic acid, 2000 mg/L Myo-inositol, 80 mg/L Niacin, 40 mg/L p-Aminobenzoate, 80 mg/L Pyridoxine, 40 mg/L Riboflavin and 80 mg/L Thiamine. When stated, the medium contained additional amino acids which consisted of 600 mg/L Alanine, 600 mg/L Arginine, 600 mg/L Cysteine, 3000 mg/L Glutamic acid, 2000 mg/L Lysine, 600 mg/L Methionine, 1500 mg/L Phenylalanine, 600 mg/L Proline, 10000 mg/L Serine, 900 mg/L Tyrosine, 4500 mg/L Valine, 2000 mg/L Aspartic acid, 4000 mg/L Threonine, 600 mg/L Histidine and 600 mg/L Tryptophan.

Example 16

Cloning of His8-yEGFP-TEV-AQPS From Milnesium tardigradum in a Bioreactor With Saccharomyces cerevisiae

[0090] A His8-yEGFP-TEV-AQPS construct was prepared following the procedure outlined in Example 14. The AQPS protein from M. tardigradum (UNIPROT: G5CTG2) was codon optimized for expression in E. coli despite the need for expression in yeast.

Example 17

Purification of His8-yEGFP-TEV-AqpZ and His8-yEGFP-TEV-AQP5 From S. cerevisiae

[0091] The purification of heterologously expressed membrane protein from S. cerevisiae was carried out as described in Example 2, except that applied buffer volumes were scaled down to match the reduced culture volumes and that lysis was carried out at 1800 bar. Protein production and purity was successfully confirmed for both fusion proteins with SDS page and Western Blot and no contaminating proteins could be detected.