Technical Field of the Invention

The present invention relates to a method for preparing closed membranes, a method for preparing closed membranes comprising target membrane proteins, as well as the closed membranes and target membrane proteins obtainable by such methods.

Background Art

Membrane proteins are involved in a large number of essential cellular processes, such as signaling, transport, energy conversion, and adhesion. They are therefore key targets for pharmaceuticals, and more than half of all drugs today are directed against membrane proteins.

In order to study membrane protein structure and function, a common approach is to overexpress the membrane protein in suitable host cells. However, many problems are associated with membrane protein overexpression, and the low yields usually obtained are a limiting factor for functional and structural studies. Common experimental problems when overexpressing foreign membrane proteins in prokaryotic microorganisms are that the overexpressed proteins become toxic to the cell, lack of proper chaperones and shortage of translocons, lack of enough membrane space for receiving the inserted proteins (the membrane gets "crowded"), formation of inclusion bodies and aggregates, and lack of suitable membrane lipids. These problems could explain why only a few percent of the solved structures in the Protein Data Bank are membrane proteins, although membrane proteins are predicted to constitute up to 30% of the encoded proteins in most genomes. A substantial effort is put in to increasing the yields of membrane proteins, but still today there is no generally applicable solution to the problem of low expression levels.

To further understand membrane protein function, it is often important to study the membrane itself and the membrane proteins in their native environment. Examples of such studies include studies of antibacterial and cell-penetrating peptides and chemicals, antibiotics, transport studies across the membrane, and coupled translation/translocation studies of proteins. Usually, either artificial liposomes of lipids (synthetic or from bacterial/animal/plant sources) or microsomes extracted from animals are used for such studies. However, such artificial membranes or membrane models may be difficult and/or expensive to produce, and there are often limited possibilities to vary the lipid composition of the membranes. Thus, the prior art fails both to provide suitable methods for preparing membranes or membrane models and to provide suitable methods for expressing target membrane proteins in cells.

Summary of the Invention It is an object of the present invention to provide a method for producing closed membranes.

A further object of the invention is to provide closed membranes suitable for different applications, such as membrane transport studies of molecules.

An object of the invention is also to provide a method for expressing target membrane proteins in closed membranes.

A further object is to provide membrane proteins.

Another object is to provide transfectionally competent cells suitable for expressing membrane proteins.

The above-mentioned objects, as well as other objects of the invention which can be gathered by a person skilled in the art after having studied the description below, are met by the different aspects of the disclosed invention.

As a first aspect of the invention, there is provided a method for preparing at least one closed membrane, comprising the steps of: a) providing a cell comprising at least one nucleic acid molecule encoding at least one protein having the ability to cause bending of at least one membrane in the cell; b) subjecting the cell to conditions such that the at least one protein having the ability to cause bending of at least one membrane in the cell is expressed in an amount sufficient to induce formation of at least one intracellular membrane in the cell; and c) disintegrating the cell comprising at least one intracellular membrane to obtain at least one closed membrane.

A "closed membrane" refers to a continuous layer of amphiphilic molecules. The closed membrane may be a lipid bilayer, which may comprise phospholipids and any other lipid, such as glycolipids. The phospholipids may be of the same or different kinds. The closed membrane may also comprise other molecules, such as proteins and/or carbohydrates. A "nucleic acid" refers to a molecule comprising monomeric nucleotides. The nucleic acid may be selected from deoxyribonucleic acid (DNA), ribonucleic acid (RNA), peptide nucleic acid (PNA), morpholino and locked nucleic acid (LNA), glycol nucleic acid (GNA) and threose nucleic acid (TNA) and mixtures thereof. The nucleic acid may encode genetic information, such as one or several proteins.

A "membrane in the cell" may be any type of membrane, such as the plasma membrane surrounding the cytoplasm of the cell or any intracellular membrane, such as a membrane of an organelle. In the context of the present disclosure, "a protein having the ability to cause bending of at least one membrane in a cell" refers to a protein that, when expressed in a sufficient amount, indirectly or directly induces bending of at least one membrane in the cell.

A protein having the ability to cause bending of at least one membrane in a cell may be defined as a bulky membrane protein, a wedge-formed protein, an interface-interacting protein, and/or a protein stimulating membrane lipid synthesis. A protein having the ability to cause bending of at least one membrane in a cell may fulfill one or several of the above-mentioned definitions. A "bulky membrane protein" refers to a transmembrane integral protein, where the exterior part of the protein or protein complex (outside the membrane) is substantially more bulky and space-demanding (laterally) than the parts crossing and residing in the lipid bilayer. Hence, if the bulky membrane protein is expressed in a sufficient amount, physical contact between the protruding parts, in combination with more than enough ("no- contact") space for the transmembrane part, may cause a "convex" bending with respect to the protrusion, when large amounts of bulky membrane protein molecules are inserted into the membrane. Two types of bulky membrane protein are schematically illustrated in Fig. 1A. A "wedge-formed protein" refers to a transmembrane protein where the integral part (in the membrane) is much more bulky inside one of the monolayers, compared to the other monolayer, and with no or very small protruding parts. Thus, the wedge-formed protein may form effectively hydrophobic "wedges" and cause bending of the membrane when expressed in sufficient amounts. The bending of a membrane due to wedge-formed protein molecules, inserted (in parallel) more or less deeply into one monolayer, may thus follow similar rules with respect to achieving bending as when membrane lipids (and other amphiphilic molecules) effect bending and budding of membranes due to the size of the polar headgroups in relation to the bulkiness and "shape" of the hydrophobic tails. A wedge-formed protein is schematically illustrated in Fig. 1 B. An "interface-interacting protein" refers to a protein with no transmembrane parts (monotopic protein), where the interface anchoring usually is achieved by a combination of charge and hydrophobicity. If an interface-interacting protein is expressed in a sufficient amount, the bulky protein molecules protruding from one side (monolayer) of the membrane will cause bending due to contacts of these parts, as more interface-interacting protein molecules are anchored as a consequence of the strong expression. For the interface-anchoring protein, there is no contact with the opposite monolayer of the membrane, and the mere continuous insertion of large amounts of protein molecules on one side may expand the area of that monolayer substantially, thus achieving a convex bending of the membrane according to established bilayer physics rules ("the bilayer couple hypothesis"). The interface-interacting protein may be complexed with other parts of the cell, such as the ribosome, forming a bulky protein export complex that attaches to a membrane. An interface-interacting protein is schematically illustrated in Fig. 1 C.

A "protein stimulating membrane lipid synthesis" refers to a protein which stimulates the formation of lipids that are inserted into the membrane. Membrane lipid biosynthesis always takes place on one monolayer in a membrane, and lipids are distributed to the other monolayer by special transporters (or by spontaneous (but slower) "flip-flop"). If a protein stimulating membrane lipid synthesis is expressed in a sufficient amount, bending of the membrane may be achieved in several ways. A strong stimulation of lipid synthesis may either bend the bilayer due to monolayer expansion or, if transmembrane equilibrium is fast, by bending the entire membrane inwards into the cytoplasm if the lateral expansion is prevented by a surrounding obstacle, like the peptidoglycan cell wall in bacteria. Further, the protein stimulating membrane lipid synthesis may be a positively charged protein. When expressed in a sufficient amount, positively charged protein molecules stimulating membrane lipid synthesis may bind to negatively charged lipids in a membrane bilayer, thereby withdrawing the negatively charged lipids from the free pool in the bilayer. This may be a signal for increased membrane lipid biosynthesis, where a "shortage" of the minus- charged lipid is sensed and counteracted by increased synthesis. If the remaining unbound lipids in the membrane bilayer have a molecular packing shape that promotes bending, the bilayer may thus start to bend as a consequence of the expression of the protein stimulating membrane lipid synthesis.

"Subjecting the cell to conditions such that the at least one protein having the ability to cause bending of at least one membrane in the cell is expressed in an amount sufficient to induce formation of at least one intracellular membrane in the cell" refers to subjecting the cell to a proper cultivation environment in terms of pH, temperature, aeration and nutrient content, such that the protein having the ability to cause bending of at least one membrane in the cell is expressed in amounts causing bending of at least one membrane and subsequent formation of at least one membrane in the cell as described above. "Disintegrating the cell comprising at least one intracellular membrane to obtain the at least one closed membrane" refers to disrupting the cells by any method so that membranes formed inside the cell can be recovered as closed membranes. Methods used for disintegration of the cells may be any common method known to a person skilled in the art, for example disintegration of the cellular membranes by enzymes such as lysozyme or using a French press (or similar method), followed by separation on a sucrose gradient using ultra- centhfugation or two-phase partitioning.

The first aspect of the invention is based on the insight that expressing a protein having the ability to cause bending of at least one membrane in a cell in sufficient amounts causes, in the appropriate circumstances, large formation of intracellular membranes, which can be recovered as closed membranes and used in different applications. Further, by using different proteins having the ability to cause bending of at least one membrane in a cell, such as different enzymes producing different types of lipids that may be inserted into the membranes, the lipid composition of the recovered closed membranes may be varied. Hence, the first aspect of the invention allows for different types of closed membranes having different properties to be prepared. The preparation method according to this first aspect of the invention is exemplified in Examples 1 and 2 of the present disclosure. These examples illustrate the efficiency and advantages of the disclosed first aspect of the invention. In one embodiment of the first aspect, the cell is a prokaryotic cell. Prokaryotic cells are easy to culture and may be cultured in large amounts. Thus, the use of prokaryotic cells for preparing closed membranes according to the first aspect of the invention does not involve complicated methods that require animal sources, in contrast to e.g. preparation of microsomes from dog pancreas. Consequently, compared to other methods in the art, the use of prokaryotic cells for preparing closed membranes may be a cheaper and more straight-forward method.

In an embodiment of the first aspect, the prokaryotic cell is a strain of Escherichia coli (E. coli). Strains of E. coli are easy to culture at low costs, thus allowing large quantities of closed membranes to be produced. The strains of E. coli may be commercial strains, such as BL21 -AI™ and/or BL21 - Star from Invitrogen. These strains have shown to be suitable cells for producing closed membranes, as shown in Examples 1 and 2 of the present disclosure.

In another embodiment of the first aspect, the cell is a eukaryotic cell. The eukaryotic cell may be a yeast cell, such as any yeast cell that is easily transfected with nucleic acid encoding at least one protein having the ability to cause bending of at least one membrane in the yeast cell. Suitable yeast strains are known to a person skilled in the art.

In one embodiment of the first aspect, the at least one nucleic acid molecule is at least one plasmid. A plasmid refers to a nucleic acid molecule that is separate from nucleic acid of the chromosomes and is capable of replicating independently of the chromosomal nucleic acid. The plasmid may be circular and/or double stranded, and may for example comprise up to 20.000 base pairs. Using at least one plasmid is a convenient way of expressing the at least one protein having the ability to cause bending of at least one membrane in the cell, as shown in Examples 1 and 2 of the present disclosure. The at least one nucleic acid may also be at least one cosmid, i.e. a hybrid plasmid containing DNA sequences from the Lambda phage. The cosmid may comprise more base pairs compared to a plasmid, such as up to 52.000 base pairs. The at least one nucleic acid may also be a fosmid, which is similar to a cosmid but based on the bacterial F-plasmid.

In another embodiment of the first aspect, the at least one protein having the ability to cause bending of at least one membrane in the cell is expressed in an amount corresponding to at least 30 mg/l culture, such as at least 50 mg/l culture, such as at least 80 mg/l culture, such as at least 100 mg/l culture, at least 120 mg/l culture, such as at least 140 mg/l culture, such as at least 150 mg/l. In the context of the present disclosure, the amount of expressed protein in the culture is measured by spectrophotometric means, such as using a Bradford assay or measuring the absorbance at 280 nm. Methods of measuring the protein concentration in a cell culture are well- known to a person skilled in the art. If a cell expresses at least one protein having the ability to cause bending of at least one membrane in the cell in an amount corresponding to at least 30 mg/l, this may ensure that large quantities of membranes are formed in the cell, thus allowing large quantities of closed membranes to be recovered from the cells.

In another embodiment of the first aspect, the at least one protein having the ability to cause bending of at least one membrane in said cell is selected from the group consisting of E. coli G3P acyltransferase PIsB, E. col 1 ATP synthase complex, E. coli fumarate reductase complex, E. coli chemoreceptor Tsr, E. coli SRP receptor FtsY, the peptidoglycan precursor glycosyltransferase MurG, cytochrome £> ₅ and PMA2/PMA1 ATPases in Saccharomyces cerevisiae, eukaryotic BAR-domain proteins, Arabidopsis thaliana monogalactosyl diacylglycerol synthase, A. thaliana digalactosyl diacylglycerol synthase, monoglucosyl diacylglycerol synthase from Acholeplasma laidlawii (alMGS) and diglucosyl diacylglycerol synthase from Acholeplasma laidlawii (alDGS).

In a further embodiment of the first aspect, the at least one protein having the ability to cause bending of at least one membrane in the cell is selected from the group consisting of monoglucosyl diacylglycerol synthase from Acholeplasma laidlawii (alMGS) and diglucosyl diacylglycerol synthase from Acholeplasma laidlawii (alDGS). The protein alMGS is a membrane interface- associated protein that catalyses the addition of a glucose molecule to a lipid acceptor, resulting in a monoglucosyl-diacylglycerol (GIcDAG) lipid. The protein alDGS is also a membrane interface-associated protein and has similar properties as alMGS. According to the present disclosure, alMGS and alDGS exhibit membrane bending properties, leading to large formation of intracellular membranes and thus large amounts of closed membranes, as seen in Examples 1 and 2.

Yet other examples of proteins having the ability to cause bending of at least one membrane in a cell may be eukaryotic proteins involved in endo- and exocytosis, membrane traffic, organelle shape maintenance, and virus assembly. Further, the ability to cause bending of at least one membrane in a cell of the proteins may be enhanced by e.g. mutagenization.

According to an embodiment of the first aspect, the at least one closed membrane is at least one vesicle. A vesicle refers to a closed membrane structure of amphiphilic molecules enclosing a liquid core. The vesicle membrane may comprise a phospholipid bilayer and other constituents, as described for the membrane in the cell above. The vesicles may have different shapes, such as a spherical shape. Vesicles may form spontaneously, or decrease in size, as the cell is disintegrated and are suitable as targets or vehicles for other processes, such as for studying: expression of membrane proteins, effects of antibacterial and cell-penetrating peptides, antibiotics, transport of small molecules, and translation/ translocation of proteins.

In a second aspect of the invention, there is provided a method for preparing at least one closed membrane comprising at least one target membrane protein, comprising the steps of: a) providing a cell comprising at least one nucleic acid molecule encoding (i) at least one protein having the ability to cause bending of at least one membrane in the cell and (ii) at least one target membrane protein; b) subjecting the cell to conditions such that the at least one protein having the ability to cause bending of at least one membrane in the cell and the at least one target membrane protein are co-expressed, wherein the at least one protein having the ability to cause bending of at least one membrane in the cell is expressed in an amount sufficient to induce formation of at least one intracellular membrane comprising the at least one target membrane protein in the cell; and c) disintegrating the cell to obtain at least one closed membrane comprising the at least one target membrane protein. A "membrane protein" refers to a protein that is attached to or associated with a membrane of a cell, such as the plasma membrane or the membrane of an organelle. The membrane protein may be an integral protein that spans both layers of a lipid bilayer membrane (a transmembrane protein), a monotopic membrane protein that is embedded in one layer of a lipid bilayer membrane, or a peripheral membrane protein that interacts with one layer of a lipid bilayer. The membrane protein may in addition be associated with or anchored to a membrane by specific groups of the membrane protein. A "target membrane protein" refers to a specific membrane protein of interest. The target membrane protein may be a protein foreign to the cell, such as a membrane protein that is not a part of the natural proteome of the cell. The target membrane protein may thus be a membrane protein from another species than the cell.

A "closed membrane comprising at least one target membrane protein" refers to a closed membrane, wherein the membrane protein is associated with the membrane of the closed membrane.

Co-expression of the least one protein having the ability to cause bending of at least one membrane in the cell and the at least one target membrane protein refers to expression of both the protein having the ability to cause bending of at least one membrane in the cell and the at least one target membrane protein. The other terms and definitions used in connection with the second aspect of the invention are as defined in connection with the first aspect of the invention above.

The second aspect of the invention is based on the insight that large amounts of target membrane proteins can be prepared by co-expressing target membrane proteins with proteins having the ability to cause bending of at least one membrane in the cell, thereby inducing formation of membranes comprising the target membrane protein in the cell. Thus, the large amounts of membranes formed in the cell facilitate the expression of target membrane proteins. Consequently, the preparation method according to the second aspect of the invention may be very helpful when overexpressing membrane proteins that are otherwise difficult to express. Preparation of target membrane proteins according to the second aspect may be performed in a high-throughput way convenient for screening purposes. The preparation method is exemplified in Example 2, which illustrates the efficiency and advantages of the disclosed second aspect of the invention.

In one embodiment of the second aspect, the at least one nucleic acid molecule encoding at least one protein having the ability to cause bending of at least one membrane in the cell and the at least one target membrane protein is the same nucleic acid molecule. This same nucleic acid molecule may be a duet vector that facilitates coexpression of different proteins. If the same nucleic acid molecule encodes both the at least one protein having the ability to cause bending of at least one membrane in the cell and the target membrane protein, it may lead to larger amounts of proteins, since the nucleic acid molecule only has to be transcribed once to initiate production of both proteins in the cell. In another, equally possible, example, the at least one protein having the ability to cause bending of at least one membrane in the cell and the at least one target membrane protein are encoded by different nucleic acid molecules. In one embodiment of the second aspect, the cell is a prokaryotic cell.

Prokaryotic cells are easy to culture and can be cultured in large amounts. Thus, using prokaryotic cells for preparing closed membranes comprising target membrane proteins according to the second aspect of the invention may allow for preparation of large amounts of closed membranes comprising target membrane proteins.

In another embodiment of the second aspect, the prokaryotic cell is a strain of Escherichia coli (E. coli). Strains of E. coli are easy to culture at low costs, thus allowing large quantities of closed membranes comprising target membrane proteins to be produced. Strains of E. coli may be commercial strains, such as BL21 -AI™ and/or BL21 -Star from Invitrogen. These strains have shown to be suitable cells for producing closed membranes comprising target membrane proteins, as shown in Example 2 of the present disclosure. In another embodiment of the second aspect, the cell is a eukaryotic cell, such as a yeast cell. The yeast cell may be any yeast cell that is easily transfected with nucleic acid encoding at least one protein having the ability to cause bending of at least one membrane in the yeast cell and a target membrane protein. Suitable yeast strains are known to a person skilled in the art.

In one embodiment of the second aspect, the at least one nucleic acid molecule is at least one plasmid. Using at least one plasmid encoding at least one protein having the ability to cause bending of at least one membrane in a cell and a target membrane protein is a convenient way of expressing the proteins, as shown in Example 2 of the present disclosure. The at least one nucleic acid may also be at least one cosmid or at least one fosmid, as described above.

In another embodiment of the second aspect, the at least one protein having the ability to cause bending of at least one membrane in the cell is expressed in an amount corresponding to at least 30 mg/l culture, such as at least 50 mg/l culture, such as at least 80 mg/l culture, such as at least 100 mg/l culture, at least 120 mg/l culture, such as at least 140 mg/l culture, such as at least 150 mg/l. If a cell coexpresses at least one protein having the ability to cause bending of at least one membrane in the cell and at least one target membrane protein, so that the at least one protein having the ability to cause bending of at least one membrane is expressed in an amount corresponding to at least 30 mg/l, it may ensure that large quantities of membranes are formed in the cell, thus allowing large quantities of closed membranes comprising the target membrane protein to be recovered from the cells.

In another embodiment of the second aspect, the at least one protein having the ability to cause bending of at least one membrane in said cell is selected from the group consisting of E. coli G3P acyltransferase PIsB, E. coli ATP synthase complex, E. coli fumarate reductase complex, E. coli chemoreceptor Tsr, E. coli SRP receptor FtsY, the peptidoglycan precursor glycosyltransferase MurG, cytochrome £> ₅ and PMA2/PMA1 ATPases in Saccharomyces cerevisiae, eukaryotic BAR-domain proteins, Arabidopsis thaliana monogalactosyl diacylglycerol synthase, A. thaliana digalactosyl diacylglycerol synthase, monoglucosyl diacylglycerol synthase from

Acholeplasma laidlawii (alMGS) and diglucosyl diacylglycerol synthase from Acholeplasma laidlawii (alDGS).

In a further embodiment of the second aspect, the at least one protein having the ability to cause bending of at least one membrane in the cell is selected from the group consisting of monoglucosyl diacylglycerol synthase from Acholeplasma laidlawii (alMGS) and diglucosyl diacylglycerol synthase from Acholeplasma laidlawii (alDGS). These proteins have been shown to facilitate the formation of large amounts of membranes comprising target membrane proteins in cells, as seen in Example 2 of the present disclosure. Further, the alMGS and alDGS proteins are only associated with the inner portion of the cytoplasmic membrane when expressed in cells. Therefore, intracellular transport of target membrane proteins to membranes and across membranes may be facilitated in the cell, since the alMGS and the alDGS proteins do not need to utilize the Sec translocon for transport across membranes. Other conceivable proteins having the ability to cause bending of at least one membrane in a cell are those discussed in relation to the first aspect of the invention above.

According to another embodiment of the second aspect, the at least one closed membrane is at least one vesicle. Vesicles are suitable carriers for target membrane proteins, thus allowing the target membrane proteins to be used in a variety of applications. In a third aspect of the present invention, there is provided a closed membrane obtainable by the method according to the first aspect of the invention. The closed membrane may comprise a phospholipid bilayer and other constituents as described for the membrane in the cell above. The closed membrane may be in any shape or form, such as in the form of a vesicle as described above.

In a fourth aspect of the invention, there is provided the use of a closed membrane according to the third aspect of the invention for studying transport of molecules across the closed membrane. The closed membranes may also be used for studying interactions of molecules with the membrane. Further, the closed membranes may also be used in a variety of other applications, such as studying cell-penetrating peptides and chemicals, and antibacterial molecules.

In a fifth aspect of the invention, there is provided a closed membrane comprising at least one target membrane protein obtainable by the method according to the second aspect of the invention. The closed membrane may be a vesicle. Vesicles comprising target membrane proteins are easy to handle and store, thus facilitating the use of vesicles comprising target membrane proteins in various applications. In a sixth aspect of the invention, there is provided a method for producing at least one target membrane protein, comprising the steps of: a) performing the method according the second aspect of the invention to obtain at least one closed membrane comprising the at least one target membrane protein; and b) recovering the at least one target membrane protein from the at least one closed membrane.

"Recovering the at least one membrane protein from the at least one closed membrane" refers to subjecting the closed membrane to conditions such that the at least one membrane protein can be isolated or extracted. Isolation or extraction of the target membrane protein may include treating the closed membranes with any one or more of various chemicals, such as detergents, or enzymes. Extracting proteins from closed membranes is known to a person skilled in the art. The other terms and definitions used in the sixth aspect of the invention are as defined in connection with the other aspects of the invention above. In a seventh aspect of the invention, there is provided a target membrane protein obtainable by the method according to the sixth aspect of the invention.

In an eighth aspect of the invention, there is provided a transfectionally competent cell, comprising at least one nucleic acid molecule encoding at least one protein having the ability to cause bending of at least one membrane in the cell.

"A transfectionally competent cell" refers to a cell that is able to take up extracellular nucleic acids, such as DNA, from its environment. The uptake may be either natural or induced by artificial means in a laboratory. The extracellular nucleic acid may be in the form of a plasmid, cosmid and/or fosmid as described above. The other terms and definitions used in relation to the eight aspect of the invention are as described above. Thus, a transfectionally competent cell comprising at least one nucleic acid molecule encoding at least one protein having the ability to cause bending of at least one membrane in the cell may provide a useful kit that may facilitate expression of a variety of target membrane proteins through transfection of the cell with a nucleic acid encoding the target membrane protein.

In a ninth aspect of the invention, there is provided the use of at least one nucleic acid encoding at least one protein having the ability to cause bending of at least one membrane in a cell for preparing at least one membrane in a cell. The terms and definitions used in the ninth aspect of the invention are as described above. Thus, the ninth aspect of the invention provides the use of nucleic acids for preparing intracellular membranes in a cell, as shown in Examples 1 and 2 of the present disclosure.

In an embodiment of the ninth aspect, the at least one protein having the ability to cause bending of at least one membrane in said cell is selected from the group consisting of E. coli G3P acyltransferase PIsB, E. coli ATP synthase complex, E. coli fumarate reductase complex, E. coli chemoreceptor Tsr, E. coli SRP receptor FtsY, the peptidoglycan precursor glycosyltrans- ferase MurG, cytochrome £> ₅ and PMA2/PMA1 ATPases in Saccharomyces cerevisiae, eukaryotic BAR-domain proteins, Arabidopsis thaliana monogalactosyl diacylglycerol synthase, A. thaliana digalactosyl diacylglycerol synthase, monoglucosyl diacylglycerol synthase from Acholeplasma laidlawii (alMGS) and diglucosyl diacylglycerol synthase from Acholeplasma laidlawii (alDGS). In another embodiment of the ninth aspect, the protein having the ability to cause bending of at least one membrane in a cell is selected from the group consisting of monoglucosyl diacylglycerol synthase from Acholeplasma laidlawii (alMGS) and diglucosyl diacylglycerol synthase from Acholeplasma laidlawii (alDGS). The use of alMGS and alDGS has been shown to lead to the formation of large amounts of membranes in cells, as shown in Examples 1 and 2 of the present disclosure.

Brief description of the drawings

Figure 1 shows schematic models of proteins that have the ability to cause bending of at least one membrane in a cell. Figure 1A shows two examples of bulky membrane proteins with large domains outside the membrane. Figure 1 B shows a wedge-formed protein. Figure 1 C shows an interface-interacting protein.

Figure 2 shows images from sucrose density gradient centhfugation tubes. Figure 2A shows fractions of BL21 -AI™-alMGS membranes after French press disruption. Figure 2B shows fractions of BL21-AI™-alMGS membranes after lysozyme disruption. Figure 2C shows fractions of BL21- Star-alDGS membranes after French cell disruption. Figure 2D shows membrane fractions from BL21 -AI™ cells not expressing alMGS or alDGS (negative control). Abbreviations used in the figure are IM = inner membrane and OM = outer membrane.

Figure 3 shows Cryo-TEM images. Figure 3A shows an elongated E. coli cell expressing alMGS filled with intracellular closed membranes. Figure 3B shows the closed membrane fraction prepared using sucrose density gradient centhfugation.

Figure 4 shows Cryo-TEM images. Figure 4A shows an elongated E. coli cell expressing alDGS filled with intracellular closed membranes and having a continuous outer membrane. Figure 4B shows an E. coli cell expressing alDGS and having a continuous outer membrane and several inner membrane compartments. Figure 5 shows SDS-PAGE of fractions from sucrose density gradient centhfugation following expression of alMGS in E. coli. Lane (1 ) = Low molecular weight marker, lane (2) = Closed membranes, 30% layer, lane (3) = IM, 35% layer, lane (4) = IM/OM, 40% layer, lane (5) = OM, 45% layer. Abbreviations used are IM = inner membrane and OM = outer membrane.

Figure 6 shows the expression results for a selection of membrane proteins from E. coli having a cytoplasmic C-terminal, which was fused to GFP. The membrane proteins were co-expressed with alMGS (in strain BL21 - AI™-alMGS) or expressed without alMGS (in strain BL21 -Al ™). The normalized average GFP fluorescence per OD ₆ oo of at least 3 experiments is shown. The number of transmembrane (TM)-segments varied from 2-12, starting from the left and increasing.

Figure 7 shows a sucrose density gradient centrifugation tube of the

E. coli membrane protein YqiJ co-expressed with alMGS. Abbreviations used in the figure are IM = inner membrane and OM = outer membrane.

Figure 8 shows the results of a western blot analysis of YqiJ (53 kDa) sucrose density gradient centrifugation fractions. Western blot was performed with α-His antibody and alMGS corresponds to 48 kDa. Lane (1 ) = YqiJ closed membrane fraction (30%), lane (2) = YqiJ IM fraction (35-40%), lane (3) = YqiJ IM/OM fraction (40%), lane (4)-(5) YqiJ OM fraction (45%), lane (6) = Pre-stained protein standard. Abbreviations used in the figure are IM = inner membrane and OM = outer membrane.

Examples

The following non-limiting examples will further illustrate the present invention.

Example 1. Formation of intracellular membranes in Escherichia coli by the monoglucosyl diacylglycerol synthase and diglucosyl diacylglycerol synthase proteins from Acholeplasma laidlawii (alMGS and alDGS, respectively). Materials and methods

Cloning and expression hosts: The cloning of monoglucosyl diacylglycerol synthase and diglucosyl diacylglycerol synthase from Acholeplasma laidlawii (alMGS and alDGS, respectively) was performed as follows: The alMGS and alDGS genes were each individually ligated into a pET-15b vector (Novagen Inc.), containing an N-terminal Hisβ-tag followed by a thrombin cleavage site before the alMGS or alDGS insert. The antibiotic selection marker for both alMGS and alDGS was 100 μg/ml carbenicillin. The Escherichia coli strain BL21 -AI™ (Invitrogen) was used as expression host for alMGS. For alDGS, the E. coli strain BL21-Star™ (Invitrogen) was used. As a control experiment, the expression cells were used without any plasmid and with 15 μg/ml tetracycline for antibiotic selection.

Cultivation: E. coli BL21 -AI™ and BL21-Star were grown using either Terrific Broth (TB) or Luria Broth (LB) medium. Enzyme expression was started with a 1 % (vol/vol) inoculum of an overnight culture to a 250 ml culture of TB media (12 g/l bacto-tryptone (Difco), 24 g/l bacto-yeast extract (Difco), 4 ml/l glycerol, 2.3 g/l KH ₂ PO ₄ and 12.5 g/l K ₂ HPO ₄ ) supplemented with appropriate antibiotics, in 2 L baffled flasks at 37 ⁰ C with 200 rpm shaking. At OD ₆ Oo ~0.6, the cultures were transferred to 22 ⁰ C and gene expression was induced after 30 min with 0.2 % (vol/vol) L-arabinose and 1 mM IPTG for the BL21 -AI™ cells and 1 mM IPTG for BL21 -Star. After 21-22 h at 22 ⁰ C, the cells were collected by centrifugation at 6000 rpm (~4000 ^χ g), 4 ⁰ C, for 30 min. The cells were washed with 50 mM HEPES, pH 8, and then again collected by centrifugation at 6000 rpm, 4 ⁰ C, for 30 min. The pellet was stored at -80 ⁰ C until use.

Sucrose density gradient analysis: The cell pellet corresponding to -600 ODU (e.g. 30 ml of cell culture of OD ₆ oo ~ 20) was thawed and resuspended in 6 ml of a buffer consisting of 50 mM triethanolamine (TEA), 250 mM sucrose, 1 mM EDTA and 1 mM dithiothreitol (DTT) at pH 7.5 supplemented with 1 mg/ml Pefabloc® and 0.1 mg/ml DNase. The cell suspension was passed through a French press, for three cycles at 1100 psi, before the cell debris was collected at 8000 rpm, 4 ⁰ C for 20 min. The supernatant was loaded on top of a 2-step sucrose gradient composed of 1 ml 55% (wt/wt) and 5.5 ml 8.8% (wt/wt) sucrose in a buffer containing 50 mM TEA, 1 mM EDTA and 1 mM DDT at pH 7.5. The tubes were centrifuged for 2 h at 210,000 x g, 4 ⁰ C using a Beckman-Coulter SW-40 rotor. Thereafter, the membrane fraction was collected from the top of the 55% sucrose layer using a syringe. The membrane fraction was loaded on top of a 6-step sucrose gradient, consisting of, from bottom to top, 0.8 ml 55%, 1.2 ml 50%, 2.0 ml 45%, 2.5 ml 40%, 1.5 ml 35% and 1.5 ml 30% sucrose in 50 mM TEA, 1 mM EDTA and 1 mM DTT, pH 7.5. The tubes where centrifuged at 4 ⁰ C, 210,000 x g, for at least 16 h using a Beckman-Coulter SW-40 rotor. The fractions were collected using a syringe and thereafter diluted to double volume with 50 mM TEA, 1 mM EDTA and 1 mM DTT, pH 7.5 before being transferred to 1.5 ml ultracenthfuge tubes. The tubes were centrifuged at 150,000 x g for 40 min at 4 ⁰ C to collect the membranes, which were dissolved in 50 mM HEPES, pH 8 and stored in -80 ⁰ C until further analyzed. The 6-step sucrose gradient tubes were photographed and the localization levels of the fractions were measured. The final fractions were analyzed using SDS-PAGE and Cryo- TEM.

Cryo-TEM was used to visualize E. coli cells and closed membranes obtained from the cells. No sectioning of the cells was performed.

SDS-PAGE analysis: Analysis was performed with NuPAGE 4-12% Bis-

Tris SDS-PAGE gels (Invitrogen) and NuPAGE MES-running buffer (Invitrogen). The SDS-PAGE gels were stained using PageBlue™ Protein Staining Solution (Fermentas) and the molecular mass marker was a low molecular weight standard (GE Healthcare).

Results - sucrose density gradient

Separation of the different membrane components was performed using sucrose density gradient centhfugation. With this technique two fractions are usually obtained: the inner membrane (IM) fraction in the 35% sucrose layer and the outer membrane (OM) fraction in the 45% sucrose layer. However, in these experiments, a lower density fraction was also obtained at the very top of the gradient or on top of the inner membrane fraction, either in the 30% sucrose layer or on top of the 30% layer. These fractions were seen in cells expressing alMGS or alDGS (Fig. 2 A-C). Cells without the plasmid expressing an enzyme, i.e. empty BL21 -AI™ cells, exhibited a normal pattern, with only two fractions at 35% and 45%, corresponding to inner membranes and outer membranes, respectively (Fig. 2D). The results showed that E. coli expressing alMGS or alDGS had large amounts of closed membranes in the cells.

Results - Cryo-TEM Cryo-TEM was used for studies of the individual fractions from the sucrose density gradient centhfugation of cells expressing alMGS and also of whole cells expressing alMGS (Fig. 3) and alDGS (Fig. 4). Most of the cells expressing alMGS and alDGS were elongated and therefore thin, and the intracellular part could be visualized (for example in Fig. 3A and 4). When looking at whole cells, it could be confirmed that there were indeed closed membranes that had formed inside the cells and totally filled the whole cytoplasma, both for cells expressing alMGS and alDGS (Fig. 3A and 4). For cells expressing alMGS, this formation of membranes in the cell could be clearly seen (Fig. 3A). Some of the closed membranes formed perfect round shapes while others formed more sausage-like shapes. The images of the closed membrane fraction from the sucrose density gradient tube showed a selection of closed membranes, most of which were shaped as vesicles and had a diameter of ~100 nm (Fig. 3B). Some closed membranes were viewed from the side and therefore visualized as thin stripes. The results from the whole-cell study of cells expressing alDGS were more or less identical to the whole-cell study of cells expressing alMGS (Fig. 4). Inner membranes filled up the cytoplasmic space, thus elongating the cells (Fig. 4A and B). Fig. 4B shows that the outer membrane stayed intact while the inner membrane divided normally and then created membranes inside the cytoplasmic compartment. Cryo-TEM was also used to investigate empty BL21 -AI™ cells, and the result showed that the cytoplasma was not filled with closed membranes but only contained a few structures, probably granules.

Hence, the Cryo-TEM images confirmed that E. coli cells expressing alMGS or alDGS had large amounts of membranes in the cells.

Results - SDS-PAGE

The fractions from sucrose density gradient centhfugation of E. coli expressing alMGS were analyzed with SDS-PAGE to study the protein distribution. alMGS (48 kDa) could be seen in all fractions, but the major part was in the fraction comprising closed membranes (Fig. 5, lane 2). The amount of alMGS in the fractions was quite high, judging from a comparison with a positive control where pure alMGS at a concentration of 0.5 mg/ml was added.

The results show that alMGS protein was present in all membrane fractions from the E. co// cells expressing alMGS.

Example 2. Expression of membrane proteins using E. co// cells expressing monoglucosyl diacylglycerol synthase from Acholeplasma laidlawii (alMGS).

Materials and methods

Membrane proteins: Twenty membrane proteins from E. co// exhibiting similar medium expression levels and number of predicted transmembrane

(TM)-segments (from two to twelve transmembrane helices) were chosen.

The proteins were YhhS, FucP, YhiV, ArcB, KdgT, YiaH, LivH, TehA, FecC, YadH, Narl, ArtM, CybB, MotA, YbbJ, YggB, YqU, YmfA, Tar and MscL. The membrane proteins selected all had a GFP (green fluorescent protein) fused to the C-terminal.

Cloning and expression hosts: The genes were amplified by PCR from the E. co// strain MG1655 and ligated into a modified pET28a (+) vector, pWaldo, which used IPTG for protein expression. The gene insert was followed by a linker with a TEV protease site, the gfp gene and a Hiss-tag at the 3 ^' end. The cloning was performed in E. co// strain MC1061. Construction of the gene for the membrane associated enzyme monoglucosyl diacylglycerol synthase from Acholeplasma laidlawii (alMGS) was performed with PCR amplification from chromosomal DNA of A. laidla wii strain A-EF22 and the gene was ligated into the pET-15b vector (Novagen Inc.) containing an N-terminal Hisβ-tag followed by a thrombin cleavage site before the alMGS insert. E. co// strain BL21 -AI™ (Invitrogen) was used as expression host for the alMGS containing plasmid. The alMGS expression cells were made competent chemically, and the resulting competent cells were denoted BL21- AI™-alMGS. Then, the selected membrane proteins of interest were transformed into the BL21 -AI™-alMGS cells. The selected membrane proteins were also transformed into normal BL21 -AI™ cells as controls. For selection of BL21-AI™-alMGS cells containing pET15b and pWaldo, 100 μg/ml carbenicillin and 50 μg/ml kanamycin were used. For the BL21 -AI™ cells containing only the pWaldo, 50 μg/ml kanamycin were used. High throughput overexpression analysis: A 24 deep-well plate was used for the analysis of expression of the selected membrane proteins. Both expression hosts, BL21-AI™ and BL21 -AI™-alMGS, were grown in parallel. The media used was Terrific Broth (TB) consisting of 12 g/l of bacto-tryptone, 24 g/l bacto-yeast extract, 4 ml/l of glycerol, 2.3 g/l KH ₂ PO ₄ and 12.5 g/l K ₂ HPO ₄ Cultures of 3 ml were inoculated with 1 % (vol/vol) overnight culture and incubated in an orbital shaker at 37 ⁰ C, 200 rpm until the cells reached OD ₆ Oo ~0.4. Cultures were transferred to 22 ⁰ C with 150 rpm shaking and induced after 30 min with 1 mM IPTG and 0.2% L-arabinose for 22 h. The amount of active protein was measured via the fluorescence from GFP. Before harvest, the final optical density at 600 nm of the cultures (OD ₆ oo ranging from 10 to 25) was measured for a fraction of the cells. The remaining cells were used for the GFP assay. These cells were harvested by centhfugation at 3500 rpm for 20 minutes and resuspended in 500 μl buffer containing 50 mM Tris-HCI (pH 8.0), 200 mM NaCI, and 15 mM EDTA. The cells were then diluted 1 :10 and 1 :20 in the above buffer to a total volume of 200 μl, which were transferred into a black 96-well Greiner plate. The remaining resuspended cells were used for e.g. western blot. The cells were incubated for 4 hours at room temperature in the dark before being analyzed for GFP fluorescence emission using a Gemini El microtiter plate reader, excitation filter 485 nm and emission filter 512 nm with a cut off of 495 nm. GFP emission from each sample was adjusted against its OD ₆ oo and the background cell fluorescence was subtracted. A normalized mean activity value for each protein was obtained from at least three independent measurements.

Sucrose density gradient analysis: A 250 ml culture of TB was inoculated with 1 % (vol/vol) overnight culture. The cells were grown at 37 ⁰ C and with 200 rpm shaking until OD ₆ oo reached -0.4. The culture was moved to 23 ⁰ C and induced after 30 min with 1 mM IPTG and 0.2 % arabinose. The protein expression was induced for 22 h before the cells were harvested at 5000 rpm for 20 min at 4 ⁰ C. OD ₆ oo was measured before harvest. The cell pellets were washed with 50 ml of 50 mM HEPES (pH 8) and then pelleted again. The cell pellet was stored at -80 ⁰ C until use. The cell pellet, corresponding to 1000 ODU, was resuspended in 6 ml buffer K (50 mM TEA, 250 mM sucrose, 1 mM EDTA and 1 mM DTT, pH 7.5) supplemented with 1 mg/ml Pefabloc® and 0.1 mg/ml of DNase. The cell suspensions were passed through a French press, three cycles, at 1100 psi. The cell debris was removed by centrifugation at 8000 rpm at 4 ⁰ C for 20 min. The supernatant suspensions were distributed on top of two-step gradients composed of 1 ml of 55 % sucrose and 5.5 ml of 8.8 % sucrose in buffer M (50 mM TEA, 1 mM EDTA, and 1 mM DTT, pH 7.5) and centhfuged for 2 hours at 210 000 x g with an SW-40 rotor (Beckman and Coulter). Thereafter, the membrane fraction was collected from the top of the 55% sucrose layer using a syringe. The membrane fraction was loaded on top of a 6-step sucrose gradient, consisting of, from bottom to top, 0.8 ml 55%, 1.2 ml 50%, 2.0 ml 45%, 2.5 ml 40%, 1.5 ml 35% and 1.5 ml 30% sucrose in buffer M. The tubes where centrifuged at 4 ⁰ C, 210,000 x g, for at least 16 h using a Beckman-Coulter SW-40 rotor. All fractions were collected using a syringe and thereafter diluted to double volume with buffer M before being transferred to 1.5 ml ultracentrifuge tubes. The tubes were centrifuged at 150,000 x g for 40 min at 4 ⁰ C to collect the membranes, which were then dissolved in 200 μl 50 mM HEPES, pH 8 and stored in -80 ⁰ C until further analyzed. The 6-step sucrose gradient tubes were photographed and the localization level of the fractions measured. The final fractions were analyzed e.g. by western blot.

Western blot analysis: Samples were diluted ten times with 50 mM HEPES (pH 8) before addition of SDS sample buffer. The samples were loaded onto a NuPAGE® 4-12% Bis Tris gel (Invitrogen) and NuPAGE® MES SDS running buffer (Invitrogen) was used. The proteins were transferred to a nitrocellulose membrane, which thereafter was blocked with 5% powder-milk and 0.1 % Tween-20 in PBS. Primary antibody was a mouse anti-penta-His antibody monoclonal IgG antibody (Qiagen) diluted 1 :5000. The secondary antibody was an anti-mouse IgG antibody, HRP conjugate (Novagen, USA), diluted 1 :5000. The membrane was developed with an ECL Plus Western blotting detection system (GE Healthcare) and detected by a LAS-1000 Plus instrument (Fujifilm).

Results - Overexpression of E. coli membrane proteins The results of the co-expression of the E. coli membrane proteins together with alMGS showed that the yield of the membrane proteins increased. Of the twenty proteins investigated, ten gave higher yields of expressed protein (Fig. 6). The protein giving the highest expression in BL21- AI™-alMGS was YqU, and the expressed amount of YqU in BL21 -AI™- alMGS was much higher compared the expressed amount of YqU BL21-AI™ cells (Fig. 6).

The results show that membrane proteins could be efficiently co- expressed with alMGS in E. coli.

Results - Sucrose density gradient

Sucrose density gradient analysis was performed on a few highly expressing proteins. In the first sucrose density separation step, a large amount of membranes were obtained (data not shown). Due to the large amount of membranes, the extracted fraction was denser and thicker and had a higher volume than normal. For the proteins exhibiting a high expression together with alMGS, a green, fluorescent fraction comprising closed membranes was obtained. The membrane protein YqU had a very high expression level together with alMGS, and green bands were seen in both 30% and 35%-40% sucrose layer, corresponding to closed membrane and inner membrane fractions (Fig. 7).

The results show that the E. coli fraction comprising closed membranes comprised large amounts of membrane protein.

Results - Western blot analysis

Fig. 8 shows western blot analysis of sucrose density gradient fractions in which the membrane protein YqU was co-expressed with alMGS. Since YqU is only slightly larger than alMGS (53 vs. 48 kDa), the band was quite difficult to see. However, in lane 1 and 2 of the western blot, two bands were seen which corresponded to the correct size (Fig. 8). These bands showed the fractions for the closed membranes and inner membranes. In the outer membrane fractions, only alMGS was monitored.

The results show that the YqU protein was present in the closed membrane and inner membrane fractions from the E. coli cells when co- expressed with alMGS.