WO2012004357A1 - Membrane comprenant des aquaporines constitutivement ouvertes - Google Patents

Membrane comprenant des aquaporines constitutivement ouvertes Download PDF

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Publication number
WO2012004357A1
WO2012004357A1 PCT/EP2011/061539 EP2011061539W WO2012004357A1 WO 2012004357 A1 WO2012004357 A1 WO 2012004357A1 EP 2011061539 W EP2011061539 W EP 2011061539W WO 2012004357 A1 WO2012004357 A1 WO 2012004357A1
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WIPO (PCT)
Prior art keywords
seq
membrane
aquaporin
water
aquaporins
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PCT/EP2011/061539
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English (en)
Inventor
Per Kjellbom
Urban Johansson
Andreas Kirscht
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Hydrogene Lund Ab
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Hydrogene Lund Ab filed Critical Hydrogene Lund Ab
Priority to EP11733640.4A priority Critical patent/EP2590732A1/fr
Priority to JP2013517393A priority patent/JP5912113B2/ja
Priority to SG2013000161A priority patent/SG186897A1/en
Priority to DK11733640.4T priority patent/DK2590732T1/da
Publication of WO2012004357A1 publication Critical patent/WO2012004357A1/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D69/00—Semi-permeable membranes for separation processes or apparatus characterised by their form, structure or properties; Manufacturing processes specially adapted therefor
    • B01D69/14—Dynamic membranes
    • B01D69/141—Heterogeneous membranes, e.g. containing dispersed material; Mixed matrix membranes
    • B01D69/142—Heterogeneous membranes, e.g. containing dispersed material; Mixed matrix membranes with "carriers"
    • B01D69/144—Heterogeneous membranes, e.g. containing dispersed material; Mixed matrix membranes with "carriers" containing embedded or bound biomolecules
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F1/00—Treatment of water, waste water, or sewage
    • C02F1/44—Treatment of water, waste water, or sewage by dialysis, osmosis or reverse osmosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants

Definitions

  • the present invention relates to a constitutively open aquaporin. Further, it relates to a membrane comprising such an aquaporin and a use of such membrane to purify water. The present invention also relates to a method of converting aquaporins, to constitutively open aquaporins.
  • Naturally occuring biological membranes have only limited intrinsic water permeability cells maintain the flux of water into and out of the cell via a family of water-specific, membrane protein channels called aquaporins.
  • aquaporin family are well known in literature, and found in archea, eubacteria and eukaryotes, including fungi, animals and plants. They serve an astonishing variety of physiological functions and are easily identified by sequence similarity across all kingdoms of life. In higher eukaryotes, water transport activity of aquaporins is frequently regulated by phosphorylation, pH and osmolarity. Aquaporins in plants and animals are highly conserved and form large protein families with 35 members in higher plants and 13 members in humans.
  • plant aquaporins are further divided into four subfamilies and their presence in primitive plants such as the bryophyte
  • PIPs plasma membrane aquaporins
  • the present invention preferably seeks to mitigate, alleviate or eliminate one or more of the above-identified deficiencies in the art and disadvantages singly or in any combination and solves at least the above mentioned problems by providing a device, a method and a use, according to the appended patent claims.
  • the general solution according to the invention is to utilize constitutively open aquaporins in a synthetic membrane.
  • a membrane comprising a constitutively open His-tagged aquaporin is provided.
  • the constitutively open His-tagged aquaporin may have at least 90 %, such as at least 95, 97 or 99%, sequence homology with SEQ ID NO: 2.
  • the membrane may comprise a constitutively open His-tagged aquaporin having amino acid sequence according to SEQ ID NO: 2.
  • the aquaporin of the membrane originates from Spinacia oleracea, Arabidopsis thaliana, Zea mays, Oryza sativa or Physcomitrella patens.
  • the aquaporin may comprise a sequence having at least 90 %, such as at least 95, 97 or 99%, sequence homology with any of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, or SEQ ID NO: 30.
  • the aquaporin may comprise a C-terminal His-tag having at least 90 %, such as at least 95, 97 or 99%>, sequence homology with SEQ ID NO: 3.
  • the aquaporin comprises the sequence according to SEQ ID NO: 1
  • SEQ ID NO: 1 SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, or SEQ ID NO: 30, and a C-terminal His-tag having according to SEQ ID NO: 3.
  • a device for purifying water comprises an inlet connected to a first chamber, for receiving dirty water, and an outlet connected to a second chamber for dispensing clean water, and a water purifying membrane comprising the membrane according to according to the first aspect, arranged in between the first and the second chamber.
  • a third aspect of the invention use of the membrane according to the first aspect or a device according to the second aspect, for purifying water is provided.
  • a method for producing a membrane according to the first aspect comprising the steps of: producing a His-tagged aquaporin, which is constitutively open; and incorporating the aquaporin into a membrane.
  • the present invention has the advantage over the prior art that it offers increased water permeability without negatively affecting the water purification.
  • Fig. 1 is a graph according to an embodiment of the invention.
  • Aquaporins e.g. SoPIP2;l (SEQ ID NO: 1)
  • SoPIP2;l SEQ ID NO: 1
  • SoPIP2;l (SEQ ID NO: 1) belong to one of the best characterized classes of aquaporins, the PIPs.
  • PIPs are known to be gated by phosphorylation of serine amino acids causing the channel to open and by protonation of a histidine amino acid or by binding of Ca 2+ causing the channel to close (Tornroth-Horsefield et al. Nature 439, 688-694, 2006).
  • the cDNA encoding AtTIP2; 1 was successfully cloned and the protein was overexpressed in Pichia pastoris with a N-terminal His-tag followed by a TEV protease site to allow removal of the affinity tag if neccesary. After opimization of the solubilization and purification the yield of AtTIP2;l corresponded to about one third of SoPIP2;l (SEQ ID NO: 1).
  • AtTIP2;l is water permeable when reconstituted in proteo liposomes, but quantitative comparison of the water transport indicated that the permeability of AtTIP2;l is approximately two thirds of the permeability of SoPIP2;l (SEQ ID NO: 1). In contrast the thermal stability of AtTIP2;l is much higher, with a melting point of 86° C compared to 57° C for So-PIP2; 1.
  • SoPIP2;l (SEQ ID NO: 1) also named PM28A was the first aquaporin shown to be gated by phosphorylations (Johansson et al. Plant Cell 10, 451-459, 1998). Later on it was shown that PIPs are also gated by pH and Ca 2+ (Tournaire-Roux et al. Nature. 425, 393-397, 2003; Alleva et al. J Exp Bot, 57, 609-621, 2006) and recently this was also demonstrated on purified AtPIP2;l reconstituted in proteoliposomes (Verdoucq et al. Biochem J, 415, 409-416, 2008).
  • SoPIP2;l aquaporin was overexpressed and purified as described by Karlsson et al. FEBS Lett 537, 68-72, 2003.
  • a P. pastoris expression kit including vectors and host cells, was purchased from Invitrogen.
  • the pm28a cDNA (GenBank accession number L77969) was amplified with the forward primer EcoRI- YPM28 A and the reverse primer Xbal- PM28A.
  • the PCR product was cloned into the P. pastoris vector pPICZB and the resulting plasmid pHT-PM28A-PICZ was sequenced.
  • the 22 amino acids added to the C-terminus of HT-PM28A are LEQKLISEEDLNSAVDHHHHHH and contain beside the 6xHis tag a myc antibody epitope.
  • a second construct was made using the forward primer EcoRI- YPM28 A and the reverse primer PM28A-REV.
  • the reverse primer has the original stop codon after PM28A and a BgUl restriction site.
  • the PCR product was cloned into pPICZB and the resulting plasmid pPM28A
  • Cell pellets were thawed and resuspended in breaking buffer (50 mM sodium phosphate buffer, pH 7.5, 5% glycerol). The cells were disrupted by three passages through a French pressure cell at 16 000 psi and lysates were clarified by centrifugation at 3000 rpm for 30 min. Membranes were collected by centrifugation at 200 OOOxg for 120 min at 4°C, resuspended in breaking buffer and stored at -80°C. Between 400 and 500 mg of crude membrane protein per liter of cell culture, or about 13 mg/g of cells, was routinely obtained.
  • breaking buffer 50 mM sodium phosphate buffer, pH 7.5, 5% glycerol
  • Peripheral membrane proteins and proteins adhering to the membranes were removed by urea/alkali treatment (such as described in Fotiadis, D., Jeno, P., Mini, T., Wirtz, S., Muller, S.A., Fraysse, L., Kjellbom, P. and Engel, A. (2001) J. Biol. Chem. 276, 1707- 1714).
  • the stripped membranes were resuspended in 20 mM HEPES-NaOH, pH 7.8, 50 mM NaCl, 10% glycerol, 2 mM L-mercaptoethanol (buffer A) at a protein concentration of about 10 mg/ml and stored frozen at -80°C until further use.
  • Stripped membranes containing 20 mg of protein were diluted to a protein concentration of 2 mg/ml and solubilized in 3% octyl-P- D -thioglucopyranoside (OTG) by dropwise addition from a stock solution of 10% OTG in buffer A. After 30 min at room temperature, unsolubilized material was pelleted at 160 OOOxg for 30 min.
  • OTG octyl-P- D -thioglucopyranoside
  • Solubilized protein was mixed for 2 h at 4°C with 2 ml of Ni-NTA agarose slurry (Qiagen) preequilibrated with 20 niM HEPES-NaOH, pH 7.8, 300 niM NaCl, 10% glycerol, 2 mM ⁇ -mercaptoethanol, 0.4% OTG (buffer B) containing 10 mM imidazole.
  • the non-bound protein fraction was removed by centrifugation and the Ni-NTA agarose was washed with buffer B+30 mM imidazole.
  • the proteins were eluted with 1 ml of buffer B+300 mM imidazole by mixing for 1 h at 4°C. Four additional elution steps were performed.
  • the detergent used for solubilisation of SoPIP2;l may be changed from OTG to OG, to avoid potential problems associated with precipitation of the detergent at low temperatures. More importantly, the Hepes buffer with imidazole may be exchanged for PBS at pH 7.5 to allow CD measurements and biotinylation of the protein and 10% of glycerol was added and recommended in all samples that were going to be frozen.
  • the overall loss of SoPIP2;l (SEQ ID NO: 1) in the extra steps added to the purification scheme was estimated to around 25%. This have not constituted a major problem since large amounts of starting material have been available but the extra step and the loss obviously resulted in an increase of the work required to deliver the same amount of protein. Incorporation of aquaporins into membranes
  • a water purification application An ocean energy application (salinity gradient energy) and an application for industrial wastewater reclamation and reuse.
  • any kind of membrane suitable for purifying water such as comprising a biological membrane, lipid membrane etc. with incorporated aquaporins, such as disclosed in US 7,857,978 B2, US
  • a method for producing a membrane comprising the steps of: producing a His-tagged aquaporin, which is constitutively open; and incorporating the aquaporin into a membrane.
  • SoPIP2; 1 A high stability of the aquaporin in the biomimetic membrane is desirable since a denaturation of the protein would compromise the permeability of the membrane.
  • the thermal stability of SoPIP2; 1 have been investigated using Circular Dichroism spectroscopy and SDS-PAGE to follow the denaturation and aggregation. As mentioned above, the melting temperature of solubilized SoPIP2;l was lower than the extremely stable AtTIP2;l . However, in proteoliposome membranes the stability of SoPIP2;l is increased to above 70°C. Preliminary data also indicate that the thermal stability is higher at high pH relative to low pH.
  • Aquaporin proteins are functioning as water-specific channels in cell membranes. Genes encoding aquaporin proteins have been inserted in a specific yeast strain and the aquaporin protein produced and purified in its functional form and used for constructing the water- specific membranes.
  • the gene coding for the spinach plasma membrane aquaporin SoPIP2;l (SEQ ID NO: 1) has been cloned into the yeast Pichia pastoris, overexpressed and purified. Without buffer exchange the yield is about 20-30 mg pure SoPIP2;l per preparation.
  • SoPIP2;l with a C-terminal His-tag (SEQ ID NO: 2) has recently been shown to be constitutively open.
  • a constitutively open His-tagged aquaporin having at least 90 %, such as at least 95, 97 or 99%, sequence homology with SEQ ID NO: 2 is provided.
  • Another embodiment relates to a constitutively open aquaporin having amino acid sequence according to SEQ ID NO: 2.
  • AtTIP2;l As described above, the plant aquaporin AtTIP2;l have been overexpressed and purified in high yields. AtTIP2;l was shown to be an active water channel when reconstituted in proteo-liposomes. AtTIP2;l is most likely a constitutively open isoform according to measurements in proteoliposomes.
  • Aquaporins from Zea mays, Oryza sativa or Physcomitrella patens may also be useful for the purpose of the invention.
  • PIPs plasma membrane aquaporins
  • the constitutively open conformation of aquaporin is achieved when the C-terminal His-tag prevents interaction between Ser274 of aquaporin, and the next monomer which can be seen in a closed conformation, depending on the exact type of aquaporin.
  • Ser274 is however conserved in all aquaporins belonging to the PIP2 group, both in higher plants and more primitive plants.
  • a number of PIP2 sequences from Spinacia oleracea,
  • Arabidopsis thaliana, Zea mays, Oryza sativa or Physcomitrella patens are provided as SEQ ID NO: 4 to SEQ ID NO: 30.
  • Another embodiment relates to a method of obtaining constitutively open aquaporin.
  • Said method comprises the step of adding a sequence having at least 90 %, such as at least 95, 97 or 99%, sequence homology with SEQ ID NO: 3, to the C- terminus of a plasma membrane aquaporin.
  • said plasma membrane aquaporin belongs to the phylogenetic group PIP2.
  • Another embodiment relates to a constitutively open aquaporin comprising a C-terminal His-tag having at least 90 %, such as at least 95, 97 or 99%, sequence homology with SEQ ID NO: 3.
  • Another embodiment relates to a membrane comprising a constitutively open aquaporin as disclosed herein.
  • sequence homology i.e. sequence identity
  • sequence identity is intended to mean the alignment sequence homology obtained by use of the BLAST- algorithm using default settings.
  • the membrane according to embodiments of the invention is incorporated into a device for purifying water.
  • Said device comprises an inlet connected to a first chamber, for receiving dirty water, and an outlet connected to a second chamber for dispensing clean water.
  • a water purifying membrane comprising the membrane according to embodiments of the invention, is arranged in between the first and the second chamber, so that water may pass from the first to the second chamber.
  • the properties of the water purifying membrane and the membrane will be easily appreciated by a person skilled in the art, such as in view of US 7,857,978 B2, US 2009/0007555 Al, EP 1 885 477 Bl, or Helix Nielsen, Anal Bioanal Chem (2009) 395:697-718.
  • use of the membrane according to embodiments or the device, for purifying water is provided.
  • the purifying may be according to a method, comprising the steps of:

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
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  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Dispersion Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Environmental & Geological Engineering (AREA)
  • Engineering & Computer Science (AREA)
  • Hydrology & Water Resources (AREA)
  • Chemical Kinetics & Catalysis (AREA)
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  • Water Supply & Treatment (AREA)
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Abstract

La présente invention concerne une membrane comprenant une aquaporine constitutivement ouverte dotée de capacités améliorées de purification d'eau. De plus, elle concerne un appareil, l'utilisation et le procédé associés à une telle membrane.
PCT/EP2011/061539 2010-07-08 2011-07-07 Membrane comprenant des aquaporines constitutivement ouvertes WO2012004357A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
EP11733640.4A EP2590732A1 (fr) 2010-07-08 2011-07-07 Membrane comprenant des aquaporines constitutivement ouvertes
JP2013517393A JP5912113B2 (ja) 2010-07-08 2011-07-07 恒常的に開いたアクアポリンを含む膜
SG2013000161A SG186897A1 (en) 2010-07-08 2011-07-07 Membrane comprising constitutively open aquaporins
DK11733640.4T DK2590732T1 (da) 2010-07-08 2011-07-07 Membran omfattende konstitutivt åbne aquaporiner

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SE1050751-5 2010-07-08
SE1050751 2010-07-08

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WO2012004357A1 true WO2012004357A1 (fr) 2012-01-12

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090007555A1 (en) 2005-09-20 2009-01-08 Peter Holme Jensen Biomimetic Water Membrane Comprising Aquaporins Used in the Production of Salinity Power
WO2009076174A1 (fr) * 2007-12-05 2009-06-18 The Board Of Trustees Of The University Of Illinois Membranes polymères hautement perméables
EP1885477B1 (fr) 2005-05-20 2010-02-17 Aquaporin APS Membrane de filtrage de l'eau

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IL166509A0 (en) * 2002-07-29 2006-01-15 Mt Technologies Inc Biomimetic membranes

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1885477B1 (fr) 2005-05-20 2010-02-17 Aquaporin APS Membrane de filtrage de l'eau
US7857978B2 (en) 2005-05-20 2010-12-28 Aquaporin A/S Membrane for filtering of water
US20090007555A1 (en) 2005-09-20 2009-01-08 Peter Holme Jensen Biomimetic Water Membrane Comprising Aquaporins Used in the Production of Salinity Power
WO2009076174A1 (fr) * 2007-12-05 2009-06-18 The Board Of Trustees Of The University Of Illinois Membranes polymères hautement perméables

Non-Patent Citations (15)

* Cited by examiner, † Cited by third party
Title
"Membrane Transport", BIOPHYSICAL JOURNAL, BIOPHYSICAL SOCIETY, US, vol. 94, no. 2, 1 February 2008 (2008-02-01), pages 245 - 255, XP025963398, ISSN: 0006-3495, [retrieved on 20080201], DOI: 10.1016/S0006-3495(08)79027-1 *
ALLEVA ET AL., J EXP BOT, vol. 57, 2006, pages 609 - 621
FOTIADIS, D., JENO, P., MINI, T., WIRTZ, S., MULLER, S.A., FRAYSSE, L., KJELLBOM, P., ENGEL, A., J. BIOL. CHEM., vol. 276, 2001, pages 1707 - 1714
HELIX NIELSEN, ANAL BIOANAL CHEM, vol. 395, 2009, pages 697 - 718
HIMANSHU KHANDELIA ET AL: "To Gate or Not To Gate: Using Molecular Dynamics Simulations To Morph Gated Plant Aquaporins into Constitutively Open Conformations", THE JOURNAL OF PHYSICAL CHEMISTRY B, vol. 113, no. 15, 16 April 2009 (2009-04-16), pages 5239 - 5244, XP055008019, ISSN: 1520-6106, DOI: 10.1021/jp809152c *
JOHANSSON ET AL., PLANT CELL, vol. 10, 1998, pages 451 - 459
KARLSSON ET AL., FEBS LETT, vol. 537, 2003, pages 68 - 72
KARLSSON M ET AL: "Reconstitution of water channel function of an aquaporin overexpressed and purified from Pichia pastoris", FEBS LETTERS, ELSEVIER, AMSTERDAM, NL, vol. 537, no. 1-3, 27 February 2003 (2003-02-27), pages 68 - 72, XP004411980, ISSN: 0014-5793, DOI: 10.1016/S0014-5793(03)00082-6 *
KUKULSKI ET AL., J MOL BIOL, vol. 350, 2005, pages 611 - 616
M. KUMAR ET AL: "From the Cover: Highly permeable polymeric membranes based on the incorporation of the functional water channel protein Aquaporin Z", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, vol. 104, no. 52, 26 December 2007 (2007-12-26), pages 20719 - 20724, XP055007842, ISSN: 0027-8424, DOI: 10.1073/pnas.0708762104 *
S. M. SAPAROV: "Water and Ion Permeation of Aquaporin-1 in Planar Lipid Bilayers. MAJOR DIFFERENCES IN STRUCTURAL DETERMINANTS AND STOICHIOMETRY", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 276, no. 34, 17 August 2001 (2001-08-17), pages 31515 - 31520, XP055008014, ISSN: 0021-9258, DOI: 10.1074/jbc.M104267200 *
See also references of EP2590732A1 *
TÖRNROTH-HORSEFIELD ET AL., NATURE, vol. 439, 2006, pages 688 - 694
TOURNAIRE-ROUX ET AL., NATURE, vol. 425, 2003, pages 393 - 397
VERDOUCQ ET AL., BIOCHEM J, vol. 415, 2008, pages 409 - 416

Also Published As

Publication number Publication date
EP2590732A1 (fr) 2013-05-15
SG186897A1 (en) 2013-02-28
JP2013535312A (ja) 2013-09-12
DK2590732T1 (da) 2015-06-22
JP5912113B2 (ja) 2016-04-27
SG10201505266QA (en) 2015-08-28

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