RU2500684C2 - Membrane protein renaturation method - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: renatured membrane protein obtaining method is proposed. The above method involves production of a homogeneous solution containing a denatured membrane protein, a detergent mixture, phospholipide or phospholipide mixture and apolipoprotein or its equivalent with: further removal of detergents and formation of lipid-protein nanodiscs (LPND) containing renatured protein.

EFFECT: method provides production of renatured membrane proteins with high yield, which are built into LPND, which can be used at development of new medical products, biocatalysts, biosensors and biophotonic devices.

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Description

The invention relates to molecular biology and biotechnology, and in particular to a method of renaturation of membrane proteins that can be used in the preparation of new drugs, the creation of new biocatalysts, biosensors and biophotonic devices.

According to current data, membrane protein (MB) genes account for up to a third of all sequences encoded in the human genome [E. Wallin, G. von Heijne, (1998) Protein Sci., 7: 1029-1038]. These proteins play a major role in signal transduction processes, both at the level of an individual cell and at the level of the whole organism, and also perform transport, energy, and other functions. Membrane proteins determine the functioning of the mammalian nervous, immune and endocrine systems. According to current forecasts, approximately half of all human membrane proteins are potential targets for the development of new drugs. The most interesting objects for pharmacological developments are two classes of MB. The first class includes membrane receptors associated with ion channels that transmit signals through or along the cell membrane by changing the permeability of the membrane [F. Hucho, C. Weise, (2001), Angewandte Chemie International Edition, 40 (17): 3100-3116]. Most of these MBs have a subunit organization and are homo- or hetero-oligomers. The second class of pharmacologically important MBs include membrane receptors associated with G-proteins (GPCR), which activate the intracellular cascade of reactions in response to extracellular signals of a chemical or light nature [D.M. Rosenbaum, S.G. Rasmussen, B.K. Kobilka, (2009) Nature, 459: 356-363]. GPCR family receptor molecules often form oligomers in cell membranes, however, the monomer consisting of 7 transmembrane helices is a functionally active unit of these MBs.

In addition to research in the field of pharmacology, MBs are of great interest as objects for biotechnological developments. Membrane enzymes responsible for the catalytic transformation of lipids in a living cell and involved in the consumption of alkanes by certain microorganisms can be used in the chemical and food industries, as well as in the field of cosmetology, to create modified molecules based on natural and synthetic lipids and hydrocarbons. Membrane enzymes and receptors can also be used in biotechnology and ecology to create nanoscale biosensors and biocatalysts capable of detecting contaminants of an alkyl nature and transforming them into biodegradable derivatives. In addition, membrane receptors that respond to light stimuli (for example, bacteriorhodopsin halobacteria) can be used in the construction of various bionanophotonic devices for storing and transmitting information.

Despite the great diversity and enormous practical importance of membrane proteins, progress in their research and application is extremely difficult. The complexity of the study and practical application of MB is associated both with the difficulty of their development and isolation, and with the features of their spatial organization. Most MBs are bundles of several transmembrane helices. Since transmembrane helices are hydrophobic, a membrane protein can have a natural structure only in the presence of a biological membrane or a suitable membrane mimetic. Moreover, the relatively weak interhelix interaction in membrane proteins causes their greater conformational mobility. Both of these factors (the requirement for a membrane and dynamic mobility) significantly complicate both the isolation and further use of MB for applied or research purposes.

To stabilize MB in solution, membrane mimetics such as detergent micelles, lipid-detergent bicelles, and lipid vesicles are usually used. These membrane mimetics have a number of disadvantages and in some cases cannot ensure the long-term stability of MB preparations. So, lipid vesicles, although they provide a quasinative environment of a protein molecule, are prone to spontaneous aggregation (fusion) with the formation of large monolamellar liposomes, which often leads to aggregation of MB preparations. In addition, lipid vesicles isolate the cytoplasmic part of MB from an external solution, which complicates functional studies and the use of MB in the field of biotechnology. On the other hand, the large surface curvature of the micelles of detergents and lipid-detergent bicelles often distorts the spatial structure of the encapsulated protein molecule, which leads to a decrease in its activity, and the high dynamic mobility of the detergent molecules significantly reduces the stability of MB. Detergent micelles and lipid-detergent bicelles are prone to dissociation when the detergent concentration is lower than the critical micelle concentration, which can also lead to inactivation of the MB drug. In addition, a molecule or MB molecules can spontaneously leave micelles or bicelles, and also move between them, which leads to the decomposition of functionally active oligomeric complexes or to an undesirable association of membrane molecules.

An alternative membrane-modeling medium is lipoprotein particles, also called reconstructed immature particles of high density lipoproteins (rHDL) or lipid-protein nanodiscs (LBD). Each lipid-protein nanodisk is a fragment of a bilayer membrane formed from phospholipid molecules, the hydrophobic part of which is screened from the solvent by one or more copies of the protein molecule apolipoprotein (Apo-LP), or its synthetic analogue (Figure 1). LBN has a disk shape with a thickness equal to the thickness of a bilayer membrane (4-5 nm). Lipoprotein particles can have a diameter of from 8 to 100 nm. The diameter of the LBN depends on the type of apolipoprotein used, the type of phospholipid and the molar ratio apolipoprotein / phospholipid used in the reconstruction of the LBN [I.G. Denisov, Y.V. Grinkova, A.A. Lazarides, S.G. Sligar, (2004) J. Am. Chem. Soc. 126 (11): 3477-3487]. For the formation of LBN, examples of the use of apoliproteins from various organisms, including mammals (humans), are described [S.E. Matz, A. Jonas, (1982) J. Biol. Chem., 257: 4535-4540.], Arthropods (silkworm B. mori) [V.A. Chromy, E. Arroyo, C.D. Blanchette, G. Bench, H. Benner, J.A. Cappuccio, M.A. Coleman, P.T. Henderson, A.K. Hinz, E.A. Kuhn, J.B. Pesavento, B.W. Segeike, T.A. Sulchek, T. Tarasow, V.L. Walsworth, P.D. Hoeprich, (2007) J Am Chem Soc, 129 (46); 14348-14354] and fish (zebrafish, Danio rerio) [S. Banerjee, T. Huber, T.P. Sakmar, (2008) J Mol Biol., 377 (4): 1067-1081], as well as apolipoprotein fragments or synthetic protein sequences including one or more copies of apolipoprotein fragments with inserts or divisions [I.G. Denisov, Y.V. Grinkova, A.A. Lazarides, S.G. Sligar, (2004) J. Am. Chem. Soc. 126 (11): 3477-3487]. In addition, synthetic peptide molecules capable of forming an amphiphilic helical structure in the presence of phospholipid molecules and stabilizing nanosized fragments of a bilayer lipid membrane in solution are used for the formation of LBN [V.K. Mishra, G.M. Anantharamaiah, J.P. Segrest, M.N. Palgunachari, M. Chaddha, S.W. Sham, N.R. Krishna, (2006) J Biol Chem., 281 (10): 6511-6519]. Unlike traditionally used micelles and bicelles, LBNs contain fragments of a bilayer membrane, which makes it possible to better simulate the membrane environment of embedded proteins. A fragment of the lipid membrane in the composition of LBN retains many of the biophysical properties inherent in these membrane systems. The main advantage of LBN is their stability. Unlike lipid vesicles and micelles of detergents, they are not prone to aggregation and fusion and do not decompose when the concentration is lowered. In addition, the protein molecule or molecules embedded in the LBN have a fixed aggregation state. Membrane proteins cannot spontaneously leave LBN, which effectively suppresses the undesired association of membrane molecules and prevents the breakdown of functionally important protein complexes. Full accessibility to the solvent on both sides of LBN (and, accordingly, the membrane protein integrated into it) allows us to carry out functional studies of MB and use MB in the field of biotechnology.

For molecular biological studies of protein molecules and for their use in the field of pharmacology or biotechnology, in most cases, large quantities of a functionally active protein preparation are required that cannot be obtained from natural sources. For the production of MB drugs, several methods are currently used with significant drawbacks. For example, heterologous expression, during which the target protein accumulates in the membrane of bacterial, yeast, insect or mammalian cells, often allows MB to be obtained in a functionally active form. However, the amount of protein preparation obtained by this method is limited by the capacity of the membranes of expressing cells and is often not enough for pharmacological and structural studies, as well as for the applied use of MB [K.N. Lundstrom (2006) Structural Genomics on Membrane Proteins. CRC Press]. Moreover, successful heterologous expression of MB in the composition of bacterial cell membranes is described only for a few eukaryotic proteins, which indicates the limited applicability of this approach [E.C. McCusker, S.E. Bane, M.A. O'Malley, A.S. Robinson, (2007) Biotechnol. Prog., 23 (3): 540-547].

Milligram quantities of MB preparations can be obtained by the methods of biotechnology or bioorganic chemistry in a functionally inactive form. For example, effective bacterial systems for the expression of membrane proteins make it possible to obtain the required amounts of MB in the composition of insoluble inclusion bodies characterized by improper packing of the target protein polypeptide chain and / or its incorrect aggregation state [E.C. McCusker, S.E. Bane, M.A. O'Malley, A.S. Robinson, (2007) Biotechnol. Prog., 23 (3): 540-547]. In addition, MB preparations can be obtained using highly effective methods of cell-free synthesis [B. Schneider, F. Junge, V.A. Shirokov, F. Durst, D. Schwarz, V. Dutsch, F. Bemhard, (2010) Methods Mol. Biol., 601: 165-186], or chemical synthesis methods [A.G. Komarov, K.M. Linn, J.J. Devereaux, F.I. Valiyaveetil. (2009) ACS Chem Biol., 4 (12): 1029-1038]. In order to convert the MB preparation obtained by these methods into the active form, an in vitro renaturation process is required using membrane mimetics such as detergent micelles, lipid-detergent bicelles, or lipid vesicles [N. Kiefer, (2003), Biochim. Biophys. Acta 1610: 57-62]. To date, there are no universal methods for renaturation of membrane proteins. Of particular difficulty is the renaturation of oligomeric and polytopic (consisting of several transmembrane helices) membrane proteins, which include most receptors and ion channels.

A known method of renaturation of membrane proteins obtained from inclusion bodies by successive replacement of detergents in the micellar environment of membrane proteins [Patent EP No. 1359155, MKL. C07K 1/113, publ. 0511.2003]. However, this approach has serious limitations in the case of renaturation of oligomeric membrane proteins as well. as in the case of micellar systems, intermolecular protein-protein contacts are difficult. As a consequence, oligomerization efficiency decreases.

A known method of renaturation of a membrane protein KcsA from the bacterium Streptomyces lividans in the native tetrameric form by direct incorporation of a biosynthetic protein into phospholipid vesicles [A. van Dalen, S. Hegger, J.A. Killian, B. de Kruijff, (2002), Febs Lett. 525: 33-38]. However, this method is laborious and the yield of the tetramer does not exceed 30%.

A known method of renaturation of membrane proteins by obtaining a homogeneous solution of membrane protein and phospholipids in a mixture of organic solvents, or a mixture of an organic solvent and water, followed by removal of solvents and complete hydration of the obtained phospholipid-protein mixture [RF Patent for the invention No. 2306319, priority 07.12.2005] . However, this approach has serious limitations in the case of renaturation of polytopic membrane proteins containing more than four transmembrane helices. Solutions of organic solvents are isotropic in their characteristics and poorly model the properties of a biological membrane with hydrophobic and polar regions. As a result of this, an abnormal formation of helix-helix contacts can occur in the membrane protein, which reduces the efficiency of renaturation.

Also known is a renaturation method based on the incorporation of membrane proteins into artificial lipid vesicles [F.I. Valiyaveetil, Y. Zhou, R. MacKinnon, (2002), Biochemistry 41: 10771-10777]. The method includes the steps of solubilizing the denatured protein with a detergent, forming mixed protein / detergent / phospholipid bicelles, removing the detergent with dialysis, and producing artificial vesicles containing the target protein. The disadvantage of this method is the inability to control the aggregation state of the protein. The resulting lipid vesicles do not protect the MB from undesirable aggregation, which can lead to significant losses of the target protein during renaturation, as well as to a significant decrease in the stability of the obtained renatured preparations. In addition, for functional studies or the use of MB in the field of biotechnology, an additional transfer (re-solubilization) of the renatured drug from lipid vesicles to other membrane mimetics, such as micelles, bicelles, or LBN, is necessary. An additional stage of resolubilization can also lead to loss of protein preparation and to a decrease in the overall efficiency of the renaturation process. When applying this approach to renature the KcsA membrane protein, the yield of the native tetrameric form of the protein is up to 30% of the initial amount of the target protein.

Closest to the claimed is a renaturation method based on the inclusion of membrane proteins solubilized in detergent sodium dodecyl sulfate (SDS) in lipid-protein nanodisks [Shenkarev 3.0. "Lipid-protein nanodisks is a universal medium for stabilization, renaturation and structural studies of membrane proteins and membrane-active peptides." International conference on biomolecular science in honor of the 75 ^th anniversary of the birth of professor Yuri Ovchinnikov, 09/28/02/2009 Abstracts, Volume 2. Young scientists competition, Moscow-Pushchino, pp. 267-268, Biochemistry 41: 10771-10777 ]. The method includes the steps of solubilizing the denatured protein in SDS, adding human lipid and apolipoprotein A1 molecules to the solubilized protein, and removing SDS using a special sorbent. As a result of the removal of SDS, the membrane protein is inserted into the lipid-protein nanodisks in a functionally active form. The disadvantage of this method is the need to use high concentrations of SDS (which by its properties refers to hard denaturing detergents [GG Prive, (2007), Methods 41: 388-397]) to solubilize all components of the reaction mixture and obtain a homogeneous solution including a membrane protein, apolipoprotein and phospholipids. The low efficiency of SDS detergent removal using special sorbents, dialysis or gel filtration leads to the loss of the protein preparation and to the incorrect formation of the spatial structure of the protein during LBN reconstruction. These factors significantly reduce the effectiveness of renaturation. When applying this approach to renature a KcsA membrane protein, the yield of the native tetrameric form of the protein is up to 50% of the initial amount of the target protein.

The objective of the invention is to provide a universal method of renaturation of membrane proteins obtained as a result of heterologous expression, cell-free or chemical synthesis, in high yield.

The problem is solved due to the fact that in the method for producing renatured membrane proteins, which includes obtaining a homogeneous solution containing a membrane protein in a detergent, a phospholipid or a mixture of phospholipids and apolipoprotein or its synthetic analogue, followed by removal of the detergent and the formation of lipid-protein nanodisks, a mixture of detergents is used where along with the detergent or mixture of detergents used to solubilize the denatured protein, an excess of additional etergenta or detergents, which can be effectively removed by means of special adsorbents, dialysis or gel filtration, and their properties may relate to a mild non-denaturating detergent [G.G. Prive, (2007), Methods 41: 388-397].

By apolipoproteins or their synthetic analogues are meant protein or peptide molecules capable of stabilizing fragments of a bilayer lipid membrane in solution having characteristic sizes (8-100 nm), including, but not limited to, apolipoproteins, apolipophorins and their derivatives, as well as sequences of apolipoproteins containing insertions or deletions, protein sequences containing one or more copies of the sequences of one or more apolipoproteins or fragments thereof, as well as peptide moieties ekuly capable of forming amphiphilic helical structure in the presence of phospholipid molecules. For example, human apolipoproteins A1 and E4, Danio rerio fish apolipoprotein A1, B. Mori silkworm apolipophorin III, their fragments and their derivatives. Apolipoprotein molecules or their synthetic analogues can be obtained both from natural sources and by chemical synthesis methods, or by cell-free synthesis, or by heterologous expression. Synthetic analogues of apolipoproteins can include “special” polypeptide and synthetic sequences (“tags”) designed to purify apolipoprotein molecules and formed lipoprotein particles by affinity chromatography, as well as to immobilize apolipoprotein and lipoprotein particle molecules on various substrates and substrates, as well as for visualization molecules of apolipoproteins and lipoprotein particles by spectroscopy, microscopy and tomography. Such sequences may include, but are not limited to, fluorescence labels or sequences for their attachment, chelating labels, contrast agents for magnetic resonance imaging, spectroscopy and microscopy, thioreactive labels, etc.

The method is as follows:

Membrane protein is obtained by any known method, for example, as a result of cell-free synthesis [B. Schneider, F. Junge, V.A. Shirokov, F. Durst, D. Schwarz, V. Dotsch, F. Bemhard, (2010) Methods Mol. Biol., 601: 165-186], or chemical synthesis [A.G. Komarov, K.M. Linn, J.J. Devereaux, F.I. Valiyaveetil. (2009) ACS Chem Biol., 4 (12): 1029-1038] or as inclusion bodies [L.E. Petrovskaya, A.A. Shulga, O.V. Bocharova, Ya.S. Ermolyuk, E.A. Kryukova, V.V. Chupin, M.Zh.Zh. Blommers, A.S. Arseniev, M.P. Kirpichnikov, (2010), Biochemistry, 75 (7): 1001-1013], or from the membranes of a producer strain [E.S. McCusker, S.E. Bane, M.A. O'Malley, A.S. Robinson, (2007) Biotechnol. Prog., 23 (3): 540-547]. The resulting protein is solubilized using a detergent or a mixture of detergents (for example, from the group of hard detergents: SDS, sodium lauryl sarcosinate, phosphocholines with different fatty acid chains Fos-12, Fos-14, Fos-16, etc.) and other reagents (chaotropic agents, such as urea, guanidine chloride; reducing agents, such as β-mercaptoethanol, dithiotriethol, tris (2-carboxyethyl) phosphine; and the like) that promote the conversion of protein from the form of insoluble aggregates to a soluble monomeric form, possibly characterized wrong to investment polypeptide chain. A homogeneous solution of the target MB in detergent or a mixture of detergents is mixed with a solution of apolipoprotein or its synthetic analogue and a solution of phospholipid or a mixture of phospholipids containing an excess of additional detergent or detergents (for example, from the group of soft detergents: sodium cholate, octyl-β-glucoside, CHAPS, CHAPSO etc.), which can be effectively removed using special sorbents, dialysis or gel filtration. The ratio of reagents should be selected by a specialist, taking into account the known properties of known reagents, while the concentration can vary widely. Then the detergents are removed from the mixture by any known method, for example, by dialysis, or by adding a special adsorbent to the mixture, or by gel filtration (Figure 2, 3). As a result of the removal of detergents, the membrane protein is embedded in the lipoprotein particles in a functionally active form. An excess of additional detergent or detergents allows you to get a homogeneous solution containing a lower concentration of detergent or a mixture of detergents used to solubilize the denatured protein, and to achieve a more complete removal of detergents during the LBF formation process. Purification of LBNs containing a renatured (functionally active) membrane protein from empty "lipoprotein particles and other reaction products (phospholipid molecules, apolipoprotein molecules and unrenatured target MB molecules) is carried out by any known method, for example, by affinity chromatography using special polypeptide sequences ("tags") pre-sewn to a membrane protein or apolipoprotein sequence, for example, a polyhistidine sequence, or by affinity chromatography imaging using antibodies against the target MB, or by the method of affinity chromatography, using the ligands of the target MB, or by gel filtration. Membrane protein renaturation is controlled either by gel electrophoresis, or by structural analysis, or by functional tests.

The inventive method allows the renaturation of membrane proteins obtained as a result of heterologous expression or cell-free synthesis, or chemical synthesis in the denatured state, in the native form simultaneously with the incorporation of the renatured protein into lipoprotein particles with a yield of up to 90% of the initial amount of the target protein. The method allows to obtain in milligram quantities recombinant structured, functionally active membrane proteins that can be used in medicine, pharmacology, ecology, biotechnology and nanobiotechnology to create new biomedical preparations, biocatalysts, biosensors and biophotonic devices.

The invention is illustrated by graphic materials:

Figure 1 presents a diagram of the device LBN;

figure 2 - diagram of the renaturation of membrane proteins using apolipoproteins or their synthetic analogues;

figure 3 - diagram of the renaturation of oligomeric membrane proteins using apolipoproteins or their synthetic analogues;

figure 4 - gel electrophoresis of the drug KcsA before renaturation in monomeric form (lane 2) and after renaturation in tetrameric form in the composition LBN / POPC (lane 3), lane 4 - preparation KcsA in tetrameric form isolated from E. coli membranes under mild conditions with the preservation of oligomeric organization, lane 1 — protein markers of molecular masses (kDa), the arrows indicate the positions of anoAl, KcsA monomer and KcsA tetramer;

figure 5 - gel filtration profiles of the preparations KcsA / LBN / POPC and ARb2 / LBN / POPC / DOPG (3: 2). Scales of particle size (nm) and elution volume of the preparations are indicated (ml), gel filtration analysis was performed on Superdex-200 resin (GE Healthcare), Tricom 5/200 column (GE Healthcare), detection is carried out by absorption at a wavelength of 280 nm;

figure 6 - gel electrophoresis of the drug ARb2 in the composition LBN / POPC / DOPG (lane 2), lane 3 - the drug ARb2 in a mixture of Fos-12 / Fos-14 / Fos-16 / LMPG, lane 1 - protein markers of molecular weights . Arrows indicate the positions of anoA1 and ARb2;

Fig.7 - one-dimensional ¹ H NMR spectra of 0.1 mm timolol-maleate (A) and 0.09 mm LBN / POPC / DOPG (3: 2) (B) (10 mm Tris-Ac, pH 7.0 , 30 ° C). The assignment of some signals of timolol maleate and phospholipids is shown.

on Fig - fragments of one-dimensional ¹ H NMR spectra of 4 μm thymolol maleate (10 mm Tris-Ac, pH 7.0, 30 ° C) in the presence of an increasing concentration of the drug "empty" LBN / POPC / DOPG (3: 2 ) (A) and ARb2 / LBN / POPC / DOPG (3: 2) (B) (10 mM Tris-Ac, pH 7.0, 30 ° C). An increase in the concentration of LBN in solution can be observed by increasing the signal intensity of the CH ₂ lipid groups in the spectrum.

The invention is illustrated by the following examples.

Example 1

Renaturation of the K ⁺ channel of KcsA from the monomeric state to the tetrameric form using apolipoproteins and a mixture of detergents of sodium dodecyl sulfate (SDS) and sodium cholate.

The bacterial K ⁺ channel KcsA (Streptomyces lividans), having a native tetrameric organization, is isolated from the inner membrane of E. coli by extraction with mild detergent DDM (β-dodecyl maltoside), as described in [L. Heginbotham, E. Odessey, C. Miller (1997) Biochemistry, 36: 10335-10342J. The KcsA preparation is then denatured to the monomeric form by precipitation with 10% trichloroacetic acid. DDM detergent is removed by washing the KcsA precipitate three times with cold acetone. The KcsA protein precipitate is then dissolved in 1% sodium dodecyl sulfate detergent (SDS). The monomeric state of the KcsA preparation thus obtained is detected by gel electrophoresis (Figure 4). The estimated molecular weight of KcsA-His ₆ (monomer) is 18.6 kDa.

The KcsA preparation in monomeric form is mixed with a solution of the synthetic analogue of human apolipoprotein A1 (fragment 44-243, apoA1) obtained in a known manner [Z.O. Shenkarev, E.N. Lyukmanova, O.I. Solozhenkin, I.E. Gagnidze, O.V. Nekrasova, V.V. Chupin, A.A. Tagaev, Z.A. Yakimenko, T.V. Ovchinnikova, M.P. Kirpichnikov, A.S. Arsenyev. (2009) Biochemistry, 74 (7): 933-945], and a solution of POPC lipid (palmitoyl-oleyl-phosphatidylcholine) in sodium cholate detergent so that the final molar ratio KcsA / anoA1 / POPC / cholate in solution is 1: 20: 900: 1800. The final molar ratio of KcsA / SDS in solution is 1: 500. The resulting mixture was incubated for 12 hours at room temperature, then the detergents (sodium cholate and SDS) were removed using the BioBeads ™ sorbent. Purification of LBFs containing KcsA from "empty" LBFs is carried out using metal aAin chromatography using His _6- tag attached to the C-terminus of the KcsA molecule. The association of KcsA with apolipoprotein A1 and lipids is confirmed by gel filtration (Figure 5), gel electrophoresis (Figure 4) and one-dimensional NMR spectroscopy. The tetrameric state of KcsA in the LBN is detected by gel electrophoresis (Figure 4). According to densitometric and spectrophotometric analyzes, the efficiency of incorporation of KcsA into LBN is at least 90% of the initial amount of the target protein, while the sample of renatured KcsA in LBN does not contain a monomeric form of the protein. Thus, the total yield of the KcsA renaturation reaction in this way is ~ 90% of the initial amount of the target protein. The total yield of the KcsA renaturation reaction in the prototype method is ~ 50% of the initial amount of the target protein

Example 2

Renaturation of human beta2-adrenergic receptor (ARb2) using apolipoproteins and a mixture of detergents Fos-12, Fos-14, Fos-16, LMPG and sodium cholate.

A modified version of the human beta2-adrenergic receptor (ARb2) containing a deletion of the C-terminal fragment (35 kDa) is obtained by heterologous expression of the fusion construct with the Mistic protein in E. coli inclusion bodies as described in [L.E. Petrovskaya, A.A. Shulga, O.V. Bocharova, Ya.S. Ermolyuk, E.A. Kryukova, V.V. Chupin, M.Zh.Zh. Blommers, A.S. Arsenyev, M.P. Kirpichnikov, (2010), Biochemistry, 75 (7): 1001-1013]. Inclusion bodies are dissolved with 1% lauryl-sarcosine detergent in the presence of 1 mM dithiothreitol. Then, the fusion protein is hydrolyzed using the thrombin enzyme, and cleaved ARb2 using His ₁₀ -tag attached to the C-terminus of the receptor molecule is purified by metal affinity chromatography in the presence of 1% lauryl-sarcosine and 10 mM β-mercaptoethanol. Then, ARb2 was reprecipitated with 10% trichloroacetic acid. Lauryl-sarcosine detergent is removed by washing the ARb2 precipitate three times with cold acetone. Then the ARb2 protein precipitate is dissolved in 25 mM NaPi buffer (pH 7.4) containing 0.7 mM trimethyl-tris (2-carboxyethyl) phosphine, 150 mM NaCl and a 2% mixture of detergents (Fos-12 / Fos-14 / Fos-16 / LMPG in a ratio of 1: 6: 1: 2). The monomeric state of the ARb2 preparation thus obtained is detected by gel electrophoresis (Figure 6, lane 3).

The drug ARb2 in monomeric form is mixed with a solution of the synthetic analogue of human apolipoprotein A1 (fragment 44-243) obtained in a known manner [Z.O. Shenkarev, E.N. Lyukmanova, O.I. Solozhenkin, I.E. Gagnidze, O.V. Nekrasova, V.V. Chupin, A.A. Tagaev, Z.A. Yakimenko, T.V. Ovchinnikova, M.P. Kirpichnikov, A.S. Arsenyev. (2009) Biochemistry 74 (7): 933-945], and a POPC / DOPG lipid solution (palmitoyl-oleyl-phosphatidylcholine / dioleyl-phosphatidylcholine 3: 2) in sodium cholate detergent so that the final molar ratio ARb2 / apoA1 / lipids / cholate in solution is 1: 20: 1600: 3200. The final molar ratio of ARb2 / Fos-12 / Fos-14 / Fos-16 / LMPG in solution is 1: 50: 300: 50: 100. The resulting mixture was incubated for 12 hours at 4 ° C, then the detergents (sodium cholate, Fos-12, Fos-14, Fos-16 and LMPG) were removed using the BioBeads ™ sorbent. The purification of LBNs containing ARb2 from “empty” LBNs was carried out using metal affinity chromatography using His _10- tag attached to the C-terminus of the ARb2 molecule. The association of ARb2 with apoA1 and lipids is confirmed by gel filtration (Figure 5), gel electrophoresis (Figure 6) and one-dimensional NMR spectroscopy.

The activity of the obtained drug ARb2 / LBN / POPC / DOPG was checked by NMR spectroscopy using the selective ligand of the human timolol beta2-adrenergic receptor. Timolol is used in the form of a salt of maleic acid (Lens). One-dimensional ¹ H NMR spectra of timolol maleate and empty LBN / POPC / DOPG (3: 2) in Tris-Ac buffer (pH 7.0) are shown in Fig. 7. A comparison of the spectra shows that the signal of the tertbutyl group of timolol (1.37 ppm) does not overlap with lipid signals from LBI. To detect the activity of renatured ARb2 in complex with LBN / POPC / DOPG, a solution (24 μM) of ARb2 / LBN / POPC / DOPG in the same buffer is gradually added to a solution of timolol (3 μM) in a Tris-Ac buffer (pH 7.0) up to final concentration of complexes ARb2 / LBN 3 μm. In the one-dimensional NMR spectra, the signals of the tert-butyl group of timolol (1.37 ppm) and the signals of CH ₂ groups of lipids from LBN are observed. The spectra obtained are shown in FIG. 8B. It can be seen from the spectra that with an increase in the concentration of the ARb2 / LBN / POPC / DOPG complexes in solution, the signal of the tert-butyl group of timolol gradually broadens and shifts to a weak field, which indicates the presence of interaction between timolol and ARb2 / LBN / POPC / DOPG. As a control, titration of a solution of timolol (4 μM) in Tris-Ac buffer (pH 7.0) with a solution of empty LBN / POPC / DOPG (50 μM) to a final LBN concentration of 5 μM is titrated. The spectra obtained are shown in FIG. 8A. It can be seen from the spectra that timolol does not interact with "empty" lipoprotein particles.

According to densitometric and spectrophotometric analysis, as well as NMR spectroscopy data, the total yield of the ARb2 renaturation reaction in this way is ~ 10% of the initial amount of the target protein. The output of the renatured ARb2 in the prototype method is not more than 2% of the initial amount of the target protein.

Claims (1)

A method of obtaining a renatured membrane protein, comprising obtaining a homogeneous solution containing a denatured membrane protein, a mixture of detergents, a phospholipid or a mixture of phospholipids and apolipoprotein or its synthetic analogue, followed by removal of detergents and the formation of lipid-protein nanodiscs containing the renatured protein.

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