JP2005225796A - Method for producing protein by using liposome - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method effective for the synthesis and recovery of a membrane protein possessing biological functions (being in a native state) in a cell-free protein synthesis system and to provide a new method for easily separating and purifying only the objective protein from the expressed protein. <P>SOLUTION: A protein expressed in a cell-free protein synthesis system and being interactive with liposomes is brought into contact with the liposomes, and a protein possessing biological functions is recovered from the resulting liposomes. The protein interactive with the liposomes can be utilized as e.g., a membrane protein or a protein fused to a tag containing a membrane protein binding site. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

The present invention relates to a method for producing a protein using liposome by means of cell-free protein synthesis. Specifically, the present invention relates to a method for producing a protein comprising a step in which a liposome is allowed to coexist in a cell-free protein synthesis system and a protein that interacts with the liposome expressed by the cell-free protein synthesis system is brought into contact with the liposome.

  Membrane proteins are not only difficult to purify because of their hydrophobic properties, but the "function" of "substance, information and energy exchange" through the membrane is said to catalyze the usual "biochemical reaction". It is difficult to measure compared to the enzymatic reaction of soluble protein, and it is very difficult to clarify the true function / activity of membrane protein.

Membrane proteins are roughly classified into the following superficial proteins and endogenous proteins.
(A) Membrane superficial protein (extrinsic protein, peripheral protein): Does not enter the hydrophobic part of the lipid bilayer, and is non-covalently bound (ionic bond, hydrophobic bond, or other) to the hydrophilic part of the lipid or other membrane protein Are weakly coupled). It can be separated from the membrane by a relatively mild extraction method such as increasing the salt concentration or treating with EDTA.
(B) Intrinsic protein, integral membrane protein: Most membrane proteins are of this type, and in most cases, the hydrophilic part protrudes on both sides of the membrane, and within the membrane, the hydrophobic part of the lipid bilayer It is a transmembrane protein that interacts. These membrane proteins, like lipids, are amphiphilic and contain hydrophobic amino acids. Polypeptide chains located inside the lipid bilayer often form α-helices and exist stably in a hydrophobic environment.

  On the other hand, in order to efficiently obtain various proteins required in research or medicine, a cell-free protein synthesis means is attracting attention today. Rabbit reticulocyte extract (Reticulocyte Lysate) was often used for this method. However, recently, based on the elucidation of the destabilization mechanism of wheat germ cell-free system (Wheat Germ Extract), a method of preparing a wheat germ extract having a stable and high translational activity and using the wheat germ extract was used. A high-efficiency cell-free protein synthesis system has been provided and used for various protein synthesis (Non-patent Document 1) (Patent Documents 1 to 4).

  Even using such a wheat germ cell-free protein synthesis system, mass production of activated proteins subjected to various modifications (acylation, amidation, etc.) including membrane protein and glycosylation is very difficult. This is because, unlike soluble proteins, most membrane proteins (and secretory proteins, intracellular granule proteins) require special mechanisms for their normal translational expression. Usually, the expression of membrane protein is suppressed, and even if the protein is synthesized, it is precipitated in the cell in a denatured state as an inclusion body.

  Furthermore, in the wheat germ cell-free protein synthesis system, many hydrophilic proteins involved in protein synthesis are present in the extract. Proteins synthesized in the wheat germ cell-free protein synthesis system undergo the process of synthesizing expressed proteins in an environment mixed with these. Therefore, when purifying an expressed protein, the conventional method uses a column or bead that is synthesized with a known tag (His × 6, GST, etc.) fused and specifically adsorbed to the tag. It was considered effective to carry out the purification. However, some known purification methods using tags have a problem in that some proteins present in the wheat germ extract remain after purification. This is because the protein derived from the germ that has the same adsorptiveness as each tag, the protein derived from the germ that adsorbs to the resin and beads of the carrier itself, and the buffer used for purification. It is thought to be caused by germ-derived proteins that wake up.

JP 2000-236896 JP Japanese Patent Laid-Open No. 2002-125693 JP 2002-204689 JP JP 2002-204689 JP Proc. Natl. Acad. Sci. USA, 99: 14652-14657 (2002)

  An object of the present invention is to provide an effective method for recovering a protein having a biological function (native state) in a cell-free protein synthesis system, and to provide a novel purification method for separating a target protein. .

This inventor examined the substance which can be utilized by a cell-free protein synthesis system based on the knowledge of the protein which interacts with a liposome, and the molecular mechanism of a liposome. As a result, if the liposome is brought into contact with the protein expressed in the cell-free protein synthesis system, and then the expressed protein is eluted from the liposome, it is possible to recover the protein that interacts with the liposome carrying the biological function. I found out. In particular, as a protein that interacts with the liposome, a protein in which a membrane protein and a tag including a membrane protein binding site are fused is useful.
In other words, the present invention
“1. A method for recovering a protein, comprising a step of bringing a liposome into the cell-free protein synthesis system and contacting the liposome with a protein capable of interacting with the liposome synthesized by the cell-free protein synthesis system.
2. 2. The method for recovering a protein according to item 1 above, comprising the step of recovering the liposome from the cell-free protein synthesis system.
3. 3. The method for recovering a protein according to item 1 or 2, comprising a step of eluting a protein capable of interacting with the liposome from the liposome.
4). 4. The method for recovering a membrane protein according to item 3 above, wherein the step of eluting the protein from the liposome is performed by a simple sucrose discontinuous density gradient centrifugation.
5). 5. The method according to any one of items 1 to 4, wherein the protein capable of interacting with the liposome is a membrane protein.
6). 5. The method according to any one of items 1 to 4, wherein the protein capable of interacting with the liposome is a hydrophobic tag fusion protein.
7). 7. The method according to item 6 above, which comprises the step of separating the protein and the hydrophobic tag by treating the hydrophobic tag fusion protein with endopeptidase.
8). A hydrophobic tag having a hydrophobic amino acid sequence and capable of interacting with a liposome.
9. A hydrophobic tag comprising a polypeptide or membrane protein binding site containing 5 to 50 hydrophobic amino acids.
10. A hydrophobic tag comprising a sequence selected from any one of the following:
(1) SEQ ID NO: 1 or SEQ ID NO: 2 (2) Sequence having at least about 70% amino acid sequence homology with SEQ ID NO: 1 or SEQ ID NO: 2 and carrying membrane protein binding ability (3) 10. a sequence having a mutation such as deletion, substitution, addition or insertion of one to several amino acids in the sequence (1) or (2) and carrying a membrane protein binding ability; 11. The hydrophobic tag according to any one of items 8 to 10 above, wherein an endopeptidase recognition sequence is further linked to the hydrophobic tag.
12 8. The recovery method according to item 6 or 7, wherein the hydrophobic tag of the hydrophobic tag fusion protein is selected from any one of items 8 to 11 above.
13. 13. A method for producing a protein using the recovery method according to any one of 1 to 7 and 12 above.
14 A reagent kit for cell-free protein synthesis means comprising at least one of the reagents used in the recovery method according to any one of 1 to 7 and 12 above.
15. 15. A protein prepared by the method for producing a protein according to item 13 or the reagent kit for cell-free protein synthesis means according to claim 14. "
Consists of.

In the present invention, a liposome capable of interacting with an expressed liposome, such as a membrane protein, in a native state with as little denaturation as possible by selectively allowing the liposome to coexist in a cell-free protein synthesis system for protein expression. Then, only the membrane protein can be easily recovered by separating the liposome from the solution. Furthermore, proteins that can interact with liposomes, for example, expressed proteins fused with a tag containing a membrane protein binding site, are expressed in a wheat germ cell-free system in the presence of liposomes, and endopeptidase can be used to purify only the target protein. I found out. As described above, a great technical advance has been brought about as a method for producing a protein capable of interacting with liposomes by a cell-free protein synthesis system and a method for purifying a target protein.

1) Liposomes The liposome used in the present invention is not particularly limited. Preferably, it is composed of molecules that actually constitute a biological membrane and is in a liquid crystal phase at room temperature (when a lipid bilayer is formed from a pure substance, two phases of a hard gel phase and a soft liquid crystal phase are formed depending on the temperature. It has a phase transition due to temperature change, and the living cell membrane is a mixture but is considered to be in a liquid crystal state. The size of the liposome can generally be 20 nm to 100 μm, preferably 200 nm to 10 μm, and more preferably GV that can be easily separated by centrifugation as described later. GV (Giant Vesicle) is a phospholipid bilayer vesicle having a diameter of 1 μm or more.
Liposomes are particles composed of artificial lipid membranes, and are formed as a lipid bilayer from phospholipids, glyceroglycolipids, cholesterol and the like. For the preparation, widely known methods such as a surfactant removal method, a hydration method, an ultrasonic method, a reverse phase distillation method, a freeze-thaw method, an ethanol injection method, an extrusion method, and a high-pressure emulsification method are applied. Details of the preparation of liposomes are described in JP-A-9-208599. For example, gel filtration, dialysis, ultrafiltration, etc. are generally used as the surfactant removal method.
Examples of the phospholipid used for preparing liposomes include phosphatidylcholine, phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, sphingomyelin, dicetyl phosphate, cardiolipin and lysophosphatidylcholine. These lipids may be extracted and purified from natural materials such as soybean oil or egg yolk, or may be hydrogenated and saturated with constituent fatty acids (hydrogenated phospholipids). Fatty acids such as those substituted with palmitic acid or myristic acid (diacylphosphatidylcholine, diacylphosphatidylglycerol, etc.) may also be used. Specific examples include purified egg yolk lecithin, hydrogenated purified soybean lecithin, egg yolk-derived phosphatidylcholine, dipalmitoyl phosphatidylcholine, dipalmitoylphosphatidylglycerol, distearyl phosphatidylcholine, dimyristylphosphatidylcholine, and the like.

2) Membrane protein The membrane protein of the present invention is not particularly limited. It can be adapted to various membrane proteins by adjusting the affinity with liposomes. Preferred examples for the time being include cytochrome b5 (C-terminal anchor type), cytochrome b561 and muscarinic acetylcholine receptor (several transmembrane type), P450-reductase (N-terminal single transmembrane type) and the like.
The cytochrome b5 exemplified in the present invention is composed of a polypeptide consisting of 133 amino acid residues and a heme bound to it non-covalently (molecular weight of about 16,000). A typical integral membrane protein, an amphipathic molecule consisting of a hydrophilic region containing heme (N-terminal 97 residues) and a hydrophobic region involved in membrane binding (C-terminal 35 residues) These two regions have an independent higher-order structure. Cytochrome b5 functions as a component of the microsomal electron transport system, receiving electrons from NADH via NADH-cytochrome b5 reductase and NADPH via NADPH-ferrihemoprotein reductase. Is to supply.
P450-reductase (NADPH-cytochrome P-450 reductase) is one of the flavin enzymes that constitute the microsomal electron transport system. The reducing power of NADPH is transferred to terminal oxygenases such as cytochrome P-450, heme oxygenase, squalene epoxidase and cytochrome b5. A polypeptide with a molecular weight of 70,000 to 80,000, which contains one FAD and one FMN, and can be regarded as a fusion protein of flavotoxin and ferredoxin: NADP + oxidoreductase. An amphipathic protein that retains the activity of reducing non-physiological electron acceptors such as cytochrome c, but cannot reduce cytochrome P-450 or b5 and lacks the ability to bind to the membrane.

3) Cell-free protein synthesis system As the cell-free protein synthesis system used in the present invention, widely known cell-free protein synthesis means can be used. For example, prepared according to Rapid Translation System RTS500 (Roshe Diagnostics), Proc. Natl. Acad. Sci. USA, 97: 559-564 (2000), JP 2000-236896, JP 2002-125693, JP 2002-204689 Wheat germ extract and its cell-free protein synthesis system {JP 2002-204689, Proc. Natl. Acad. Sci. USA, 99: 14652-14657 (2002)} can be used. In particular, a system using a wheat germ extract (trade name Protios) is preferable.

4) Interaction between liposome and protein prepared by cell-free protein synthesis means Liposomes are prepared in advance and before or after protein synthesis, more preferably before protein synthesis. It is added to the protein synthesis system. The amount added can be appropriately adjusted according to the amount of protein synthesized, but is generally about 0.1 to 100 mM. It is also effective to replenish liposomes as the reaction progresses. It is preferable to keep contact for about 3 to 100 hours after the addition while the cell-free protein synthesis system is active. As the temperature condition, an optimum temperature for protein synthesis of about 20 to 30 ° C is sufficient. In addition, the interaction between the liposome and the protein in the present invention means that all or part of the protein expressed in the liposome and the cell-free synthesis system is taken into the liposome. It also means the case of being localized.

5) Method for recovering membrane protein from liposome
In the recovery of membrane protein from the liposome, the cell-free protein synthesis reaction is completed, and after sufficient interaction between the liposome and the membrane protein is completed, the cell-free protein synthesis system and the liposome are separated. For the separation, general methods such as centrifugation, gel filtration, discontinuous density gradient centrifugation and the like are sufficient, and are not particularly limited. The separated liposomes are suspended in a buffer solution that is conditioned in consideration of the properties of the protein of interest. After the suspension, for example, a surfactant or the like is added to dissolve the liposome, the target protein is recovered, and the surfactant is removed later.
The following method is also useful. For example, a hydrophilic tag is attached to the end of the target membrane protein by genetic manipulation and expressed in the presence of liposomes. When a container with the antibody of the tag attached to the surface with high density is prepared, and the liposome is put therein, the liposome is trapped on the surface of the container by the adhesive action between the tag attached to the membrane protein and the antibody on the surface of the container. With the membrane protein immobilized on the interface, if the lipid molecules that make up the liposome are slowly removed using other surfactants, the membrane proteins are brought together by bringing the hydrophobic parts together, and the membrane protein further increases. Only the flake state sticking to the interface is formed. When a high concentration of a single tag molecule is added here, the equilibrium of specific adsorption shifts and the membrane protein flakes formed on the surface of the container come off. Therefore, the membrane protein flakes can exist in an aqueous solution because the upper and lower surfaces are hydrophilic, and can be recovered by allowing crystallization by concentration.

6) Novel recovery method of target protein The present invention can be used as a protein fusion capable of interacting with liposomes by binding a specific tag to a target protein to produce a tag fusion protein. The tag used for this purpose is preferably a hydrophobic tag. The hydrophobic tag in the present invention is a polypeptide containing 3 to 100, preferably 5 to 50, hydrophobic amino acids. Alternatively, it contains an amino acid sequence carrying membrane protein binding ability (membrane binding site). Furthermore, as described later, an endopeptidase recognition sequence may be linked to a part of the hydrophobic amino acid sequence. Examples of the hydrophobic tag in the present invention include those in which an endopeptidase recognition sequence is linked to several hydrophobic amino acid sequences.
As an example of a method for recovering a target protein using a hydrophobic tag, a liposome and a hydrophobic tag fusion protein are allowed to interact with each other by utilizing a portion originally present in a lipid bilayer within a cell. For example, cytochrome b5 is composed of an N-terminal part having enzyme activity and a C-terminal part having membrane binding ability. If this membrane-bound portion is used as a tag, a hydrophilic protein fused with a hydrophobic tag can be expressed and separated and purified in a wheat germ cell-free system in the presence of liposomes. In addition, the target protein-hydrophobic tag-liposome complex separated and purified by this method can be separated from the target protein and hydrophobic tag-liposome complex by the action of various known endopeptidases. Is possible.

In addition, the hydrophobic tag including a membrane protein binding site according to the present invention preferably includes SEQ ID NO: 1 which is the full-length sequence of cytochrome b5 protein or SEQ ID NO: 2 which is a partial sequence. Furthermore, in the sequence having at least about 70% amino acid sequence homology with SEQ ID NO: 1 or SEQ ID NO: 2 and carrying membrane protein binding ability, and the sequence of (1) or (2) above, 1 Hydrophobic tags including sequences having mutations such as deletion, substitution, addition or insertion of several amino acids and carrying membrane protein binding ability are also suitable.
Moreover, the hydrophobic tag containing the membrane protein binding site according to the present invention can link a sequence (endopeptidase recognition sequence) to be cleaved with endopeptidase in the sequence.
Deletion, substitution, addition or insertion means are known per se, for example, site-directed mutagenesis, gene homologous recombination, primer extension or polymerase chain amplification (PCR), alone or in combination, for example, Edited by Sambrook et al. [Molecular Cloning, Laboratory Manual Second Edition] Cold Spring Harbor Laboratory, 1989, edited by Masami Muramatsu [Lab Manual Genetic Engineering] Maruzen Co., Ltd., 1988, Ehrlich, HE. Chapter [PCR Technology, Principles and Applications of DNA Amplification] It can be carried out according to the methods described in Stockton Press, 1989, etc., or by modifying those methods. For example, Ulmer's technology (Science, 219, 666, 1983).
In the introduction of mutations as described above, from the viewpoint of carrying membrane protein binding ability, for example, homologous amino acids (polar amino acids, nonpolar amino acids, hydrophobic amino acids, hydrophilic amino acids, positively charged amino acids, negatively charged amino acids, aromatics) Mutual substitution between amino acids and the like is readily envisioned.

  The endopeptidase may be any peptidase that can cleave only the target protein from the hydrophobic tag fusion protein. For example, precision protease {endopeptidase that selectively cleaves the sequence of the precision site (Leu-Glu-Val-Leu-Phe-Gln ↓ Gly-Pro) at the position of the arrow: Amersham Biosciences}, Thrombin, Factor Xa etc. can be used.

  In the present invention, at least one of the reagents used in the protein recovery method as described above can be provided as a reagent kit for cell-free protein synthesis means. The kit provides a novel purification method capable of easily separating and purifying only the target protein from the expressed protein.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the following examples should be regarded as an aid for obtaining specific recognition of the present invention, and the scope of the present invention is limited by the following examples. Is not to be done.

1. Preparation of liposomes 10 mM solution of neutral phospholipid {specifically using DMPC (dimilystoyl-phosphatidylcholine), DOPC (dioleoyl-phosphatidylcholine) or EggPC (Egg-York Phosphatidyl Choline) as the preferred phospholipid} Add 500 mL of CHCl ₃ / MeOH = 2/1 (v / v) volume ratio mixed solvent, preferably dehydrated solvent} to the glass test tube, remove the solvent under an argon gas atmosphere, and remove the lipid film at the bottom of the glass test tube. Was prepared. The test tube was placed in a desiccator and vacuum-dried overnight using a vacuum pump to completely remove the solvent. Hereinafter, this state is referred to as “dry lipid film”. Add 500 mL of pH buffer solution (preferably HEPES buffer 100 mM, pH = 7.0) to the dried lipid film, replace the air in the test tube with argon gas, seal, and hold in a 50 ° C. water bath for 1 hour. And returned to room temperature. At this time, the solution becomes cloudy. This state is referred to as “pre-hydrated liposome”. Pre-hydrated liposomes with Buffer Mix {prepared by Wakken Pharmaceutical Co., Ltd., Invitrotech Co., Ltd. Protios (TM) supplied solution: Buffer # 1 1.07ml Buffer # 2 1.25ml MilliQ-Water 7.68ml ratio 4500 mL was gently added.
In addition, after this operation, it was confirmed by observation with a dark field microscope or a fluorescence microscope that the liposome showed a spherical structure with a diameter of several to several tens of μm. Hereinafter, this state is referred to as “liposome preparation solution”.

2. Cell-free protein synthesis system 1) Chemicals and kits used
A wheat germ-derived cell extract was used as a cell-free protein synthesis system in vitro. The wheat germ-derived cell extract used was a commercially available kit {Wakken Yakuhin Co., Ltd., Invitrotech Co., Ltd.} {Trade name: Protios (TM)}.

2) Preparation of mRNA mRNA was prepared using a gene cloned into a plasmid vector {Protis (TM) dedicated vector pEU3-NII} as a template.
In pEU3-NII (Fig. 1a), we designed a primer that introduced the fluorescent protein (GFP) gene on the 5 'side and 3' side (no stop) using Spe1 and Kpn1 sites as adapters. Amplified and introduced into MCS. Next, with respect to the fusion protein (membrane protein: cytochrome b5 in Example 1, P450 reductase in Example 2) gene, a primer having Kpn1 and BamH1 sites introduced as adapters on the 5 ′ side and 3 ′ side was designed. It was introduced into the prepared plasmid (FIG. 1b).
In addition, the fluorescent protein expression plasmid vector into which the fusion protein has not been introduced is a primer in which the gene for fluorescent protein (GFP) is introduced into pEU3-NII on the 5 ′ side and 3 ′ side (no stop) using Spe1 and Kpn1 sites as adapters. Design and use the PCR reaction
-The gene was amplified and introduced into MCS (Fig. 1c).
In addition, m-RNA was synthesized and purified as appropriate according to the instruction manual of Protios (TM).

3. Expression and recovery of cytochrome b5 protein
In an expression system solution using PROTEIOS, a liposome preparation solution was added so that the final lipid concentration was 0.5 to 12.5 mM, and synthesis was carried out by a multi-layer method (see the instruction manual of PROTEIOS). Furthermore, cytochrome b5 protein incorporating GFP was examined by microscopic observation, separation and purification by a novel sucrose discontinuous density gradient centrifugation method, and confirmation by SDSP-PAGE. Moreover, what expressed only GFP protein was used as control.

4). Protein Synthesis by Multilayer Method First, reaction mix {10 mg / ml creatine kinase 1.7 μl, Buffer # 2 2.4 μl, 40 U / μl Rnase Inhibitor 1.0 μl, 1.3 μg / μl mRNA solution <2. Depending on the preparation of mRNA> 10 μl, 200 (OD) wheat germ extract 10 μl, liposome solution 25 μl} and liposome mixed buffer mix (H ₂ O 1.34 ml, Buffer # 1 0.535 ml, Buffer # 2 0.625 ml Then, 2.5ml) of liposome solution was prepared. Next, 250 μl of the prepared liposome-containing buffer mix was placed in one hole of a 96-well plate, and 50 μl of the prepared liposome-containing reaction mix was overlaid on the lower layer. Then, translation was performed by incubating with PG-Mate ™ for 17 hours at 26 ° C. to synthesize cytochrome b5 protein incorporating GFP. Furthermore, GFP protein was synthesized as a control.

5). Fluorescence microscope observation 10 mL of a solution after expressing cytochrome b5 protein incorporating GFP in the presence of liposomes was taken to prepare a glass preparation of the solution. A fluorescence microscope equipped with a high-sensitivity camera and a high-intensity mercury lamp (eg, microscope Nikon E600 and camera Hamamatsu Photonics AQUACOSMOS), GFP filter {Nikon GFP (R) -BP, etc.} and high-sensitivity objective lens (Nikon Plan Fluor 100x, etc.) A glass preparation was prepared by an ordinary method and observed with an excitation fluorescence microscope (excitation wavelength: 460-500 nm, fluorescence wavelength 510-560 nm). At this time, for the microscopic image of the photographed sample, the ratio of the fluorescence intensity between the liposome image part and the other part is calculated using image analysis software (eg, Hamamatsu Photonics AQUACOSMOS). The localization of cytochrome b5 protein incorporating GFP was observed. Further, the position of GV was confirmed by ph contrast (phase contrast image).

6). Results of fluorescence microscope observation
The result after observing the post-expression solution of cytochrome b5 protein in which GFP is incorporated in the presence of liposomes is shown (FIG. 2). In the protein in which only GFP was expressed, nonlocal fluorescence was observed over the entire field of view regardless of the position of GV confirmed by phase contrast microscopy (ph contrast) (FIG. 2a). In the cytochrome b5 protein incorporating GFP, a structure in which a spherical outline having a diameter of several to several tens of mm emits fluorescence in green can be confirmed. This is the fluorescence of GFP incorporated in the membrane part of the liposome (FIG. 2b). For each fluorescent image, histograms were taken with the horizontal axis representing luminance (fluorescence intensity) and the vertical axis representing frequency of appearance (FIGS. 3a and 3b). In the protein that expressed only GFP, the fluorescence has almost uniform background light as one peak (Fig. 3a), but in the cytochrome b5 protein in which GFP is incorporated, a very bright fluorescent part appears as a peak. (FIG. 3b: arrow part). This indicates the localization (interaction) of cytochrome b5 protein in which GFP is incorporated in the liposome portion.

7). Examination of separation and purification by novel sucrose discontinuous density gradient centrifugation and confirmation by SDS-PAGE
A method for easily separating cytochrome b5 protein incorporating GFP from captured liposomes was examined and confirmed by SDS-PAGE.
The result of SDS-PAGE analysis of the precipitate fraction when the post-expression solution was centrifuged at 15,000 G x 15 minutes was able to separate embryo-derived proteins and synthetic proteins that were insolubilized by the synthesis reaction from liposome-synthetic protein complexes. Not. In order to separate them, the conditions of discontinuous sucrose density gradient centrifugation were examined, and the following new sucrose discontinuous density gradient centrifugation method (hereinafter referred to as “simple sucrose discontinuous density gradient centrifugation”). Developed).
Simple sucrose discontinuous sucrose density gradient centrifugation (1) A buffer containing 0.3 M sucrose (prepared by adding sucrose to Buffer Mix; hereinafter referred to as sucrose solution) was prepared.
(2) 200 μl of the sucrose solution was added to an Eppendorf tube. Subsequently, 90 μl of the protein-expressed solution was layered on the sucrose solution to form two layers, and centrifuged at 15,000 G × 15 minutes.
(3) The upper layer and the upper part of the lower layer were collected as much as possible using a thick tip and added to another new Eppendorf tube.
(4) To the collected solution, 3 times the amount of Buffer Mix was added and centrifuged at 15,000 G × 15 minutes.
(5) The supernatant was discarded and the same amount of Buffer Mix was added. Centrifuge again at 15,000 G x 15 minutes.
(6) (5) was repeated once more, and finally the supernatant was discarded and the pellet was collected.

8). Separation and purification of cytochrome b5 protein containing GFP Pretreatment (100 ° C, 5 min) with the sample pellet as it is or with 10% SDS in an appropriate amount with 10% SDS is performed, and SDS-PAGE is performed. Performed (lanes 7-11). For comparison, both 12 μl post-expression solution precipitation (lane 1) and 30 μl precipitation fraction (lanes 2-6) were used.

9. Results of separation and purification of cytochrome b5 protein incorporating GFP Conditions in each lane (in FIG. 4) are shown below.
Lane 1: 12 μl of solution after reaction (control without liposome)
Lane 2: Precipitated fraction obtained by centrifuging 15 μL of the post-reaction solution expressed in a final concentration of 1 mM liposome for 15 minutes at 15,000 G. Lane 3: Post-reaction solution of 30 μl expressed in a final concentration of 2.5 mM liposome. Precipitation fraction lane 4 centrifuged at 15,000 G for 15 minutes: 30 μl post-reaction solution expressed in final concentration 5 mM liposomes Precipitation fraction lane 5 after centrifugation at 15,000 G for 15 minutes: final concentration 7.5 mM liposomes Precipitation fraction lane obtained by centrifuging 30 μl of the post-reaction solution expressed in the medium at 15,000 G for 15 minutes: Precipitation obtained by centrifuging 30 μl of the post-reaction solution expressed in the final concentration of 10 mM liposome for 15 minutes at 15,000 G Fraction lane 7: Fraction lane purified by simple sucrose discontinuous density gradient centrifugation with 60 μl of post-reaction solution expressed in final concentration 1 mM liposome 8: Expressed in final concentration 2.5 mM liposome 60 μl of the reacted solution after purification was purified by simplified sucrose discontinuous density gradient centrifugation. Fraction lane 10 purified by simple sucrose discontinuous density gradient centrifugation: 60 μl of post-reaction solution expressed in 7.5 mM liposomal final concentration Fraction lane 11 purified by simple sucrose discontinuous density gradient centrifugation: 60 μl of post-reaction solution expressed in final concentration 10 mM liposome was purified by simple sucrose discontinuous density gradient centrifugation Fractionation

  From the electrophoresis results, it was found that the simple sucrose discontinuous density gradient centrifugation method can easily separate membrane proteins from liposomes. Furthermore, in the case of DOPC (GV), it was shown that the recovery rate was high when the protein was expressed in the presence of liposomes at 2.5 mM to 7.5 mM (final concentration).

1. Expression and recovery of P450 reductase
In the preparation of mRNA, P450 reductase was synthesized in the same manner as in Example 1 except that the membrane protein gene was mixed with the P450 reductase gene and the liposome was mixed with 5 mM in the reaction mix. Furthermore, separation and purification of P450 reductase by simple sucrose discontinuous density gradient centrifugation and confirmation by SDS-PAGE were performed.

2. As a result of separation and purification of the solution after the expression of P450 reductase From FIG. 5, it is possible to confirm the band of P450 reductase incorporating GFP in the precipitate fraction and the purified fraction, but in the purified fraction only a single band ( The arrows in the figure indicate that P450 reductase incorporating GFP has been recovered and purified.

New recovery method of target protein Determination of liposome-binding region of cytochrome b5
Based on the expression vector of the EGFP-cytochrome b5 fusion protein, a cytochrome b5-EGFP fusion protein in which the length of the hydrophilic portion sequence was variously expressed was expressed using genetic engineering techniques. The expressed fusion protein was purified by simple discontinuous density gradient centrifugation and analyzed by SDSPAGE to confirm a band in which a partial sequence of cytochrome b5 and a liposome formed a complex. The partial sequence of cytochrome b5 that formed the complex was the amino acid sequence set forth in SEQ ID NO: 2. In addition, in the full-length sequence of cytochrome b5 protein as well, a band in which a full-length cytochrome b5 sequence (SEQ ID NO: 1) and a liposome formed a complex was confirmed. Therefore, it was found that the sequence containing the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 can be used as a hydrophobic tag.

2. Recovery of soluble protein A vector comprising a precision site sequence between the hydrophobic tag sequence and the EGFP protein sequence with the full-length sequence (SEQ ID NO: 1) and partial sequence (SEQ ID NO: 2) of the obtained cytochrome b5 as a motif was constructed. . Next, the tag (cytochrome b5) -EGFP fusion protein (SEQ ID NOs: 3 and 4) was expressed in the presence of liposome in the same manner as the protein synthesis method by the multilayer method described above using the vector. Next, it is purified by simple sucrose discontinuous density gradient centrifugation, and further, EGFP is separated from the hydrophobic tag by treatment with precision protease (16h, 4 ° C), and in addition, the hydrophobic tag-liposome complex Was precipitated by centrifugation, and the supernatant was recovered. Further, the supernatant fraction was confirmed by SDS-PAGE (lane P: referred to as precision protease-treated fraction). Furthermore, for comparison, both the post-expression solution (lane T) and the fraction purified by simple sucrose discontinuous density gradient centrifugation (lane S) were used.

3. As a result of recovering soluble protein, EGFP bands can be confirmed in the fraction recovered by the simple sucrose discontinuous density gradient centrifugation method and the fraction treated with precision protease, but almost single in the fraction treated with precision protease. This indicates that only EGFP is recovered and purified.

1. Recovery of soluble protein using DHFR In the same manner as in Example 3, a vector containing a precision site sequence between a hydrophobic tag sequence (SEQ ID NO: 1) using the full-length cytochrome b5 sequence as a motif and a DHFR protein sequence was constructed. Next, using the vector, the tag (cytochrome b5) -DHFR fusion protein (SEQ ID NO: 5) was expressed in the presence of liposome in the same manner as the protein synthesis method by the multilayer method shown above. Next, it is purified by simplified sucrose discontinuous density gradient centrifugation, and further, DHFR is separated from the hydrophobic tag by treatment with precision protease (16 h, 4 ° C.), and in addition, the hydrophobic tag-liposome complex Was precipitated by centrifugation, and the supernatant was recovered. Furthermore, the supernatant fraction was confirmed by SDS-PAGE (lane P: fraction treated with precision protease). Furthermore, for comparison, both the post-expression solution (lane T) and the fraction purified by simple sucrose discontinuous density gradient centrifugation (lane S) were used.

2. Results of Soluble Protein Recovery FIG. 7 shows that the precision protease-treated fraction has only a single band, and that only DHFR is recovered and purified.

  The industrial applicability of the present invention is that proteins capable of interacting with liposomes expressed using a cell-free protein synthesis system can be easily concentrated, recovered and purified in a native state in cells.

It is explanatory drawing of exclusive vector pEU3-NII of Plotios (TM). a is a map of pEU3-NII. b shows the expression vector of cytochrome b5 or P450 reductase incorporating GFP. c shows a GFP expression vector. (Examples 1 and 2) It is the microscope image of the cytochrome b5 in which GFP was incorporated in the presence of GV and the solution after GFP expression. a is a microscope image of GFP protein, and b is a microscope image of cytochrome b5 in which GFP is incorporated. (Example 1) The histogram with respect to the fluorescence image of the cytochrome b5 in which GFP was incorporated in the presence of GV and the solution after GFP expression is shown. a shows a histogram of GFP protein, and b shows a histogram of cytochrome b5 incorporating GFP. (Example 1) It is an electrophoretic diagram of SDS PAGE after separation and purification of cytochrome b5 incorporating GFP. M represents LMW (Low molecular weight marker: manufactured by Amersham Biosciences, total 5.75 μg, detection is CBB staining; the same in the following examples). (Example 1) It is an electrophoretic diagram of SDS PAGE after separation and purification of P450 reductase incorporating GFP. M is LMW, 12T is a 12 μl post-expression solution, 30p is a 30 μl precipitate fraction, and 200 is a purified 200 μl fraction. (Example 2) It is an electrophoretic diagram of SDS PAGE of a fraction treated with precision protease. M is LMW, N is 12 μl of negative control, T is 12 μl of post-expression solution, S is a fraction purified by simple sucrose density gradient centrifugation for 60 μl, and P is a fraction treated with precision protease for 90 μl. Example 3 It is an electrophoretic diagram of SDS PAGE of a fraction treated with precision protease. M is LMW, N is 12 μl of negative control, T is 12 μl of post-expression solution, S is a fraction purified by simple sucrose density gradient centrifugation for 60 μl, and P is a fraction treated with precision protease for 150 μl. (Example 4)

Claims (15)

  A method for recovering a protein, comprising the step of bringing a liposome into the cell-free protein synthesis system and contacting the liposome with a protein capable of interacting with the liposome synthesized by the cell-free protein synthesis system.   The method for recovering a protein according to claim 1, comprising a step of recovering the liposome from the cell-free protein synthesis system.   The method for recovering a protein according to claim 1 or 2, comprising a step of eluting the protein capable of interacting with the liposome from the liposome.   The method for recovering a membrane protein according to claim 3, wherein the step of eluting the protein from the liposome is performed by a simple sucrose discontinuous density gradient centrifugation.   The recovery method according to any one of claims 1 to 4, wherein the protein capable of interacting with the liposome is a membrane protein.   The method according to any one of claims 1 to 4, wherein the protein capable of interacting with the liposome is a hydrophobic tag fusion protein.   The recovery method according to claim 6, comprising a step of separating the hydrophobic tag from the protein by treating the hydrophobic tag fusion protein with endopeptidase.   A hydrophobic tag having a hydrophobic amino acid sequence and capable of interacting with a liposome.   A hydrophobic tag comprising a polypeptide or membrane protein binding site containing 5 to 50 hydrophobic amino acids. A hydrophobic tag comprising a sequence selected from any one of the following:
(1) SEQ ID NO: 1 or SEQ ID NO: 2 (2) Sequence having at least about 70% amino acid sequence homology with SEQ ID NO: 1 or SEQ ID NO: 2 and carrying membrane protein binding ability (3) A sequence having a mutation such as deletion, substitution, addition or insertion of one to several amino acids in the sequence (1) or (2) and carrying a membrane protein binding ability   The hydrophobic tag according to any one of claims 8 to 10, wherein an endopeptidase recognition sequence is further linked to the hydrophobic tag.   The method according to claim 6 or 7, wherein the hydrophobic tag of the hydrophobic tag fusion protein is selected from any one of claims 8 to 11.   The manufacturing method of the protein using the collection | recovery method of any one of Claims 1-7, 12.   A reagent kit for cell-free protein synthesis means comprising at least one reagent used in the recovery method according to any one of claims 1 to 7 and 12.   A protein prepared by the method for producing a protein according to claim 13 or the reagent kit for cell-free protein synthesis means according to claim 14.

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