KR102473556B1 - The evaluation method for the structure of chromatophore membrane vesicle - Google Patents

Abstract

Chromatophore membrane vesicle (CMV) is a biomaterial derived from photosynthetic bacteria and has a vesicle structure similar to liposome. It can be converted into chemical energy such as H. Since ATP and NAD(P)H are coenzymes used as energy sources for biological enzymes, the use of CMV in enzyme-using industries can contribute to reducing production costs. For the industrial use of CMV, quality control of CMV is essential. Until now, the ATP synthesis activity per pigment has been used as a criterion for evaluating CMV quality. However, since this does not identify the structural characteristics of CMV, a new analysis method for quality evaluation based on the structure of CMV was needed. Since CMV has structural characteristics consisting of a membrane and a protein, the present invention utilizes a phosphatase fusion protein in which phosphatase is fused as a translational frame to a membrane protein encoding a light mechanism to prevent structural damage to CMV. A new CMV quality evaluation standard was proposed by developing a method for quantitatively analyzing the intactness and membrane orientation. As a result, it is possible to more accurately analyze and solve problems when using CMV, and the present technology can be used for purification of CMV and optimization of reaction conditions.

Description

The evaluation method for the structure of chromatophore membrane vesicle}

The present invention is a kind of photosynthetic membrane vesicle (chromatophore membrane vesicle, CMV), which is a biological material capable of converting light energy into chemical energy. It relates to a method for analyzing and evaluating intactness and orientation using a phosphatase fusion protein.

Living organisms that perform photosynthesis have a cell membrane structure in which proteins for photosynthesis are concentrated, which is called a photosynthetic membrane. There are several types of photosynthetic cell membranes, but representative ones are the thylakoid membrane (TM) present in plants, algae or cyanobacteria, and the chromatophore membrane (intracytoplasmic membrane) present in purple nonsulfur bacteria. , ICM). The photosynthetic cell membrane vesicle is a functionally independent mechanism that performs the photosynthetic light reaction and converts light energy into chemical energy such as ATP (adenosine 5'-triphosphate) and NAD(P)H (nicotinamide adenine dinucleotide (phosphate) reduced). can be converted to ATP and NAD(P)H thus formed are used as cofactors that serve as energy sources for reactions of various enzymes in vivo.

The photosynthetic membrane is purified in the form of a vesicle when separated from the living body, and it is called a photosynthetic membrane vesicle. Photosynthetic cell membrane vesicles are biomaterials that can carry out photosynthetic light reactions independently when appropriate light is irradiated in an extracellular environment, and can be used as energy converters that can convert light energy into chemical energy (registered a patent in Korea). No. 10-17087520000). By using photosynthetic cell membrane vesicles, it is possible to solve the problem of supplying high-cost coenzymes (ATP, NAD(P)H), which is one of the major limiting factors in the production of useful substances using enzymes, so it can greatly contribute to reducing production costs in the enzyme industry. Therefore, attempts are being made to produce high value-added useful substances from low-cost starting substrates by combining photosynthetic cell membrane vesicles and biological enzymes (Korean Patent Application Nos. 10-2017-0121109 and 10-2017-0052361).

Among the photosynthetic membrane vesicles, the chromatophore membrane vesicle (CMV), which is highly utilized as an ATP generator, is a complex composed of a phospholipid bilayer and a membrane protein. Since the light mechanism cell membrane performs the light reaction through electron transfer through membrane proteins and formation of a proton motive force around the membrane, the internal environment must be isolated from the external environment through a closed vesicle structure. Functional domains of membrane proteins must be placed in the correct orientation across the membrane. That is, in order for CMV to function functionally, the membrane structure must have integrity (form a completely closed structure) and the membrane must be configured such that membrane proteins are positioned in the correct direction. Therefore, if information on the structural integrity and orientation of the CMV membrane can be obtained, the ratio of functionally active vesicles in the vesicle sample can be inferred, which can be used as a quality evaluation criterion for CMV. For this reason, the present invention proposes a method that can quantitatively evaluate the quality of CMV in terms of membrane structure by using a phosphatase fusion protein in which a membrane protein encoding the light mechanism is fused with phosphatase as a translational frame. wanted to

The matters described as the background art are only for explanation of the background of the present invention, and should not be taken as an admission that they correspond to prior art already known to those skilled in the art.

CMV, a type of photosynthetic membrane vesicle, is a bio-derived photoreactor that can generate ATP by converting light energy, and is highly utilized in the enzyme industry. CMV can be purified from purple non-sulfur bacteria, which are photosynthetic bacteria, using sucrose density gradient ultracentrifuge. However, a method for evaluating the quality of purified CMV has not yet been established. Until now, measurement of the ATP synthesis activity per pigment (bacteriochlorophyll a ) of CMV has been the only criterion for evaluation. However, since this does not identify the structural characteristics of CMV, a new analysis method for quality evaluation based on the structure of CMV was needed. Accordingly, the present invention aims to develop a method for quantitatively evaluating the degree of structural damage of CMV and the directionality of the film, thereby establishing a new quality evaluation standard for CMV.

Photovesicle membrane vesicles (CMVs) are biomaterials that can synthesize ATP when light is irradiated outside the cells. CMV is a liposome structure composed of a phospholipid bilayer, and on the CMV membrane is a light-harvesting complex, a reaction center, an electron transfer chain, and ATP synthase. Membrane proteins such as are located. Since these structural characteristics of CMV are closely related to functional parts, only structurally complete CMV can function properly. However, if the CMV purification process is not appropriate or the storage and reaction conditions are not optimized, the structure of CMV may be adversely affected, which in turn results in a decrease in the function of CMV. Therefore, accurately identifying the structural state of CMV is an essential process for improving the quality of CMV.

The structure of CMV can be analyzed from two aspects. The first aspect is to analyze the degree of structural damage (intactness) of the membrane constituting the CMV. In order for CMV's ATP synthase to work, a proton motive force must be formed across the membrane. At this time, a vesicle having a completely closed structure is referred to as an intact vesicle, and a vesicle having an open structure is referred to as a leaky vesicle (see FIG. 1A ). CMV is mostly isolated in the form of intact vesicles during initial purification, but may change to the form of damaged vesicles as the membrane structure of the vesicle is disrupted by external factors. The second aspect is to analyze the orientation of CMV membranes. CMV forms a vesicle structure when purified from cells, and at this time, two types of intact vesicles (closed vesicles) can be formed depending on the direction of the membrane (see FIG. 1B). One is the inside-out vesicle, in which the periplasmic membrane faces the inside of the vesicle and the cytoplasmic membrane faces the outside of the vesicle; It is an outside-out vesicle that faces inward. In order for CMV to function normally outside the cell, it must take the form of a forward vesicle. The reason is that the functional domain of the membrane protein constituting CMV is asymmetrically arranged around the membrane, so that the membrane protein moves according to the direction of the membrane. This is because the direction of the functional domain is determined. For example, ATP synthase present in CMV has a functional region exposed in the direction of the cytoplasmic membrane within the cell, and because of this, the cytoplasmic membrane must become a forward vesicle facing outward so that the functional domain and the substrate in the reaction solution can contact. Expressed another way, it becomes a functionally effective vesicle only when the orientation of the CMV membrane is the same as that of the intracytoplasmic membrane in the original cell. As a result, only CMV with an intact inside-out vesicle form can be said to be structurally correct and functional CMV.

In the present invention, a method of fusing a phosphatase enzyme to a protein constituting CMV was used to analyze the structure of CMV (FIG. 1). Phosphatase means an enzyme that hydrolyzes a phosphoric acid monoester bond, and when para-nitrophenyl phosphate (pNPP), one of the compounds with a phosphoric acid monoester bond, is used as a substrate 420 It is easy to detect enzyme activity by forming para-nitrophenol (pNP), a substance having absorbance at nm, as a product. Therefore, the enzymatic activity of phosphatase can be used to detect a fusion protein (phosphatase fusion protein) of a protein constituting CMV and phosphatase. One of the important characteristics of pNPP, a substrate of phosphatase, is that it freely passes through the outer membrane but does not pass through the inner membrane. Because CMV consists of the photoreceptor cell membrane, which is a part of the cell's inner membrane, pNPP cannot pass through the vesicle membrane and penetrate into the interior. However, when SDS (sodium dodecyl sulfate) and CHCl ₃ (chloroform) are added to CMV, the vesicle membrane structure is destroyed, allowing pNPP to penetrate into the vesicle interior.

The C-terminus of PufA (encoded by the pufA gene of R. sphaeroides ), one of the proteins constituting the light-harvesting complex I of CMV, to prepare a phosphatase fusion protein. A fusion protein PufA-PhoA was prepared by fusing Esherichia coli- derived basic phosphatase (alkaline phosphatase, PhoA, encoded by the phoA gene of E. coli ) in the translational frame at the site (FIG. 1). PufA protein is located on the photosynthetic cell membrane in cells and is a membrane protein consisting of one transmembrane domain, with the N-terminus pointing toward the cytoplasm and the C-terminus toward the cell periplasm (Law et al. 2004. Mol. Memb. Biol. 21: 183-191). Therefore, in intact forward vesicles upon CMV purification, the PufA protein is located on the vesicle membrane with the C-terminus pointing toward the interior of the vesicle. If PhoA is fused to the C-terminus of PufA, PhoA is directed toward the inside of the vesicle in the forward vesicle and forms a structure isolated from the outside by the vesicle membrane (Fig. 1, ①). On the other hand, during CMV purification, in addition to intact forward vesicles (①), intact reverse vesicles (②) or damaged vesicles (③) may also be formed. In the reverse vesicle, the membrane structure of the forward vesicle is inverted, and when the PufA-PhoA protein is present, the PhoA portion is directed toward the outside of the vesicle. Therefore, forward vesicles do not show phosphatase activity when pNPP is injected, but reverse vesicles or damaged vesicles show activity. Here, assuming that the amount of PufA-PhoA per CMV is uniform when CMV is formed, it can be seen that the phosphatase activity by PufA-PhoA reflects the amount of CMV. When a specific CMV sample was treated with SDS and CHCl ₃ , the phosphatase activity of all CMV (T = ①+②+③) appeared, and in the untreated case, phosphatase activity by reverse vesicles and damaged vesicles (I = ②+ Since only ③) appears, the difference in phosphatase activity between the SDS/CHCl ₃ treated group and the untreated group (TI = ①) reflects the amount of forward vesicles in the sample. Therefore, calculating the ratio of total CMV to forward vesicles ((TI)/T) provides an estimate of the proportion of forward vesicles in the sample.

Based on the above concept, the present invention provides a method for analyzing the structure of a photomechanical cell membrane vesicle (CMV) comprising the following partial steps:

a) preparing a gene encoding a phosphatase fusion protein by conjugating a membrane protein gene constituting CMV with a phosphatase gene and a process of preparing a plasmid carrying the gene;

b) transforming a purple non-sulfur bacterium using the plasmid prepared in a) above, and purifying CMV containing a phosphatase fusion protein from the strain; and

c) Determining the phosphatase activity of CMV containing the phosphatase fusion protein to estimate the proportion of CMVs with the correct structure.

Also, the present invention

a) a plasmid containing a fusion gene in which a gene encoding a membrane protein present on the membrane of photoluminal cell membrane vesicle (CMV) and a gene encoding phosphatase are transcriptionally spliced; or

b) Provided is a composition for structural analysis of light instrumental cell membrane vesicles comprising the purple non-sulfur bacterial strain transformed with a) above.

“Photosynthetic membrane vesicle” means a biological material obtained by refining the photosynthetic cell membrane in the form of a vesicle, which performs the photosynthetic light reaction in a photosynthetic organism. Photosynthetic cell membrane vesicles can convert light energy into chemical energy such as ATP and NAD(P)H when irradiated with light in vitro.

The “chromatophore membrane vesicle (CMV)” is a photosynthetic membrane vesicle derived from a purple non-sulfur bacterium. Organisms that can be used as raw materials for CMV purification are Rhodobacter sp., Rhodospirillum sp., Rhodopseudomonas sp., Rhoseobacter sp., Brady It may be selected from the genus Rhizobium ( Bradyrhizobium sp.) and the genus Rubrivivax ( Rubrivivax sp.).

In a preferred embodiment of the present invention for the purpose of analyzing the membrane structure of CMV, phosphatase is fused to PufA protein, a membrane protein of CMV, but preferably all membranes that can be located on the vesicle membrane of CMV instead of PufA may be selected from among proteins. In addition, all or part of the sequence of the corresponding membrane protein can be used to prepare a phosphatase fusion protein.

The “phosphatase” refers to an enzyme that catalyzes a reaction of liberating inorganic phosphate from a substrate through hydrolysis. Specifically, water (H ₂ O) molecules are added to the phosphoric acid monoester bond of the substrate to catalyze the reaction of separating the alcohol group and phosphoric acid. There are several types of phosphatase, but in the embodiment of the present invention, alkaline phosphatase (PhoA) derived from E. coli was used. However, phosphatases that can be selected for preferred implementation are not limited to basic phosphatases.

Phosphatase can use various compounds as substrates in vitro, and the substrates are preferably selected from compounds having a phosphoric acid monoester bond. Particularly, in a preferred embodiment of the present invention, para-nitrophenyl phosphate (pNPP) is used as a substrate for phosphatase. This is because when pNPP loses its phosphate group by phosphatase, it becomes para-nitrophenol (pNP), a compound having maximum absorbance at 420 nm, which is used to easily measure the enzymatic activity of phosphatase. However, substrates for measuring phosphatase activity are not limited to pNPP, and preferably include substrates that exhibit color development, fluorescence, or luminescence by enzymatic reactions, or substrates labeled with radioactive substances. may be selected from compounds.

When phosphatase is fused to a target protein for structural analysis of CMV, all or part of the phosphatase-encoding gene sequence can be used. In addition, although phosphatase is fused to the C-terminal direction of the target protein in the embodiment of the present invention, a method of fusing to the N-terminal direction of the target protein is also included in the scope of the present invention.

The definition of the above “fusion protein” means a translatationally-fused protein that is precisely in the frame of translation. That is, each gene encoding two or more polypeptides of different origin is spliced at the gene level to have one start codon and one stop codon, which is It refers to a single peptide formed through transcription and translation. A fusion protein is a set of structural domains of each constituent protein before fusion, and each structural domain can independently perform a function, and its function may be similar to or different from the original protein before fusion.

The plasmid for inserting the gene encoding the phosphatase fusion protein is not limited, but preferably should be selected from the group of plasmids capable of replication and maintenance in purple non-sulfur bacteria such as pRK415, pIND4, and pLV106.

Previously, the activity of ATP synthase per pigment was used as the only quality evaluation criterion for CMV. However, it was insufficient to accurately analyze the quality change caused by the complex structure of CMV. In the present invention, a method for analyzing the structural state of CMV was developed to supplement and improve these existing CMV quality evaluation criteria. To this end, a criterion for quantitatively evaluating the functionally effective CMV ratio in a CMV sample was prepared by utilizing a phosphatase fusion protein. If this is used, the CMV sample state can be analyzed from various aspects, and based on this, problems that occur when using CMV can be solved from a more accurate perspective. In addition, the technology of the present invention can be used to optimize the CMV purification rate and operation efficiency through quantitative analysis of CMV quality under various conditions.

1 is a schematic diagram of a method for isolating CMV in an R. sphaeroides strain expressing PufA-PhoA (WP) and a method for estimating the ratio of intact forward vesicles.
Figure 2 shows the structures of plasmids used to express the phosphatase fusion proteins PufA-PhoA and PufA-PhoA-his ₆ in R. sphaeroides .
Figure 3 confirms the in situ activity of phosphatase (PhoA) in W-415 and WP strains cultured in Petri dishes, and obtains the strains to measure phosphatase activity. Expression of phosphatase fusion protein in cells will confirm
Figure 4 confirms that the fusion protein is contained in CMV (CMV-PH) purified from the W-PH strain through western blot using an anti-histidine-tag antibody (anti-his ₆ -tag antibody). will be.
Figure 5 shows the measurement of phosphatase activity according to SDS/CHCl ₃ treatment in purified CMV-P, and the ratio of intact forward vesicles was calculated.
Fig. 6 shows changes in total PhoA activity of CMV-P measured for one week.
Figure 7 shows the change in the proportion of intact forward vesicles in CMV samples measured for one week using CMV-P.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. Since these examples are only for explaining the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

In the following examples, a phosphatase fusion protein (PufA-PhoA) was expressed in R. sphaeroides and CMV was purified from these strains to obtain CMV containing the phosphatase fusion protein. In the process, it was confirmed that the phosphatase fusion protein was normally expressed in the strain and that the fusion protein was contained in the purified CMV. And, by measuring the phosphatase activity of purified CMV, the ratio of CMV with intact forward vesicles in the sample was calculated, and using this, the change in the ratio of intact forward vesicles in CMV samples over time under specific storage conditions was calculated. measured.

[Example 1] Preparation of plasmids and strains

In order to purify CMV containing the phosphatase fusion protein, first, a plasmid into which the phosphatase fusion protein gene was inserted was prepared (FIG. 2), and an R. sphaeroides wild type strain was transformed with the plasmid to obtain the phosphatase fusion protein. R. sphaeroides strains were prepared (Table 1). When preparing the plasmid pRK-pufA-phoA (Fig. 2A), the pufA gene was included through PCR (polymerase chain reaction) from total DNA of R. sphaeroides using primers pufF (SEQ ID NO: 1) and pufR (SEQ ID NO: 2). A gene fragment (G1) was obtained, and a phoA gene fragment (G2) was obtained through PCR from total DNA of E. coli using primers phoF (SEQ ID NO: 3) and phoR (SEQ ID NO: 4). Then, G1 was treated with HindIII and PstI , and G2 was treated with PstI and XbaI , respectively, and then G1 and G2 were simultaneously ligated to the HindIII /XbaI restriction sites of the pRK415 plasmid to pRK -pufA-phoA plasmid was prepared. When preparing the plasmid pRK-pufA-phoA-his (FIG. 2B), a gene fragment was obtained from the pRK-pufA-phoA plasmid by PCR using primers pufF (SEQ ID NO: 1) and phoR2 (SEQ ID NO: 5), and then Hin After restriction enzyme treatment with dIII and Xba I, pRK-pufA-phoA-his plasmid was prepared by conjugation to the Hin d III/Xba I restriction enzyme site of pRK415 plasmid.

name Characteristic plasmid pRK415 Tc ^r ; ori IncP Mob RP4 lacZ α
(Keen et al. 1988. Gene 70: 191-197) pRK-pufA-phoA Tc ^r ; pRK415 + PufA-PhoA fusion protein coding gene (SEQ ID NO: 6) pRK-pufA-phoA-his Tc ^r ; pRK415 + PufA-PhoA-his ₆ fusion protein coding gene (SEQ ID NO: 7) strain WT R. sphaeroides KCTC 12085, Wild type
(Lee et al. 2002. Appl. Microbiol. Biotechnol. 60: 147-153) W-415 WT + pRK415 W-P WT + pRK-pufA-phoA W-PH WT + pRK-pufA-phoA-his

R. sphaeroides wild type was transformed using the two plasmids prepared above to prepare R. sphaeroides strains WP and W-PH containing the respective plasmids pRK-pufA-phoA and pRK-pufA-phoA-his. . Additionally, a control strain, W-415, was prepared by transforming R. sphaeroides with the plasmid pKR415.

[Example 2] Purification of CMV containing phosphatase fusion protein

In order to isolate CMV, an appropriate R. sphaeroides strain was inoculated into 250 ml of Systrom's minimal medium (Sistrom. 1962. J. Gen. Microbiol. 28: 607-616, see Table 2 for composition), followed by inoculation of 15 Watt/m ² It was cultured under anaerobic conditions at 30 °C under light intensity. When the absorbance at 660 nm of the culture medium reached 2.0, centrifugation was performed at 7,000 g and 4° C. for 10 minutes to obtain a cell pellet. Subsequent purification was performed in an anaerobic chamber (model 10, Coy laboratory product) with a gas composition of 90% nitrogen, 5% hydrogen, and 5% carbon dioxide. The obtained cells were dissolved in 8 ml of disruption buffer (10 mM Tris-Cl, pH 8.0, 10% sucrose, 5 mM sodium ascorbate), and then a protease inhibitor cocktail was added according to the manufacturer's instructions. Thereafter, using a sonicator (model VCX130, Sonics & Materials), cell disruption was performed three times at 5-minute intervals, and the supernatant was separated after centrifugation at 7,000 g for 10 minutes. The supernatant was subjected to ultracentrifugation at 30,000 g and 4° C. for 2 hours, and only the membrane pellet formed at this time was obtained and dissolved again in a disruption buffer. And, in order to purify photosphere cell membrane vesicles using sucrose density gradient ultracentrifuge, 60%, 40%, and 20% sugar solutions (10 mM Tris-Cl, pH 8.0 in order from the bottom) dissolved in) layer was formed, and then the cell membrane precipitate lysate was piled up. Then, ultra-high-speed centrifugation was performed at 30,000 g and 4° C. for 4 hours to separate the reddish-brown sample (CMV) formed between the 20% and 40% sugar layers, and using 10 mM Tris-Cl (pH 8.0) buffer, 1: It was diluted in a volume ratio of 1 and stored at 4°C. CMV was indirectly quantified by measuring the amount of bacteriocholoyphll a (BChl a ). After mixing 10 μl of CMV sample and 490 μl of dye extraction solution (acetone: methanol = 7:2) and centrifuging at 6,000 g for 1 minute, absorbance at 770 nm and molar extinction coefficient of the supernatant were 83.9 The amount of BChl a was calculated using mM ^-1 (Kim and Lee. 2010. J. Bacteriol. 192: 198-207).

Systrom minimal medium additive final concentration KH ₂ PO ₄ 20 mM NaCl 8.5 mM (NH ₄ ) ₂ SO ₄ 3.78 mM L-Glutamic acid 0.67 mM L-Aspartic acid 0.25 mM Succinic acid 34 mM Nitrilotriacetic acid 1.05 mM MgCl ₂ ·6H ₂ O 1.2 mM CaCl ₂ 2H ₂ O 0.23 mM FeSO ₄ 7H ₂ O 7 µM (NH ₄ ) ₆ Mo ₇ O ₂₄ 0.16 µM EDTA 4.7 µM ZnSO ₄ 7H ₂ O 38 µM MnSO ₄ H ₂ O 9.1 µM CuSO ₄ 5H ₂ O 1.6 µM Co(NO ₃ ) ₂ 6H ₂ O 0.85 µM H ₃ B O ₃ 1.8 µM nicotinic acid 8.1 µM Thiamine hydrochloride 1.5 µM Biotin 41 nM

[Example 3] Measurement of phosphatase activity of CMV

To measure phosphatase activity in vitro, 10 μl of the sample containing phosphatase was mixed with 990 μl of buffer A (1 M Tris-Cl, pH 8.0, 0.1 mM ZnCl ₂ ). Then, 50 μl of 0.1% (w / v) SDS (sodium dodecyl sulfate) and 50 μl of CHCl ₃ were added, mixed by stirring, and left at 4 ° C. for 5 minutes. At the same time, the SDS/CHCl ₃ untreated group was left at 4° C. for 5 minutes without adding SDS/CHCl ₃ . Next, 100 μl of 0.4% (w/v) pNPP (para-nitrophenyl phosphate) was added, stirred, and reacted at 37° C. for 5 minutes, followed by 120 μl of termination buffer (0.8 M KH ₂ PO ₄ , pH 8.0, 0.1 M EDTA). After stirring, the mixture was reacted at 6,000 g and 4° C. for 5 minutes to obtain a supernatant. After measuring the 420 nm absorbance of the supernatant with a spectrophotometer (UV-2550, Shimadzu, Kyoto, Japan), the molar extinction coefficient of pNP (para-nitrophenol) was 13.2 mM ^-1 cm ^-1 (Malamy and Horecker. 1966. Methods. Enzymol. 9: 639-642) was used to determine the phosphatase enzyme activity.

[Example 4] Confirmation of expression of phosphatase fusion protein

In this Example, it was confirmed that the fusion protein was normally expressed in WP expressing the phosphatase fusion protein, and that CMV (CMV-P) purified from WP contained the fusion protein. First, in order to confirm the expression of the phosphatase fusion protein in the cell, the activity of the phosphatase enzyme was confirmed in-situ in the cells cultured in a Petri dish (FIG. 3). The phosphatase of the Puf-PhoA fusion protein expressed in WP is located in the cell periplasm. Since pNPP, a substrate of phosphatase, cannot pass through the cell inner membrane, but can freely pass through the outer cell membrane and access the cell periphery, when pNPP is added to the strain, pNP, a reaction product, is formed by phosphatase. . Since pNP has maximum absorbance at 420 nm and is yellow to the naked eye, cells that come into contact with the substrate develop a stronger yellow color than cells that do not. In the results of FIG. 3, when a 0.4% pNPP solution was treated in one area of the cells on the Petri dish (indicated by a yellow square), the WP, a strain expressing the phosphatase fusion protein, was strongly It was colored yellow. Since R. sphaeroides originally did not possess phosphatase and the color reaction occurred only in the area treated with pNPP, it can be seen that this reaction is specific color development by intracellular phosphatase. After obtaining cells of W-415 and WP and treating them with SDS/CHCl3, the phosphatase activity of the whole cell was measured. As a result, WP showed 40 times higher phosphatase activity than the control W-415 (Fig. 3). Therefore, it can be said that the phosphatase fusion protein was normally expressed in WP.

Upon CMV purification, PufA-PhoA expressed in R. sphaeroides was localized on the membrane of CMV. Therefore, if PufA-PhoA was expressed at the correct location, fusion proteins should be detected in purified CMV. To confirm this, western blot was performed, and anti _- histidine-tag antibody (anti-his ₆ -tag antibody) was detected using an immune response (FIG. 4). First, CMV-PH, CMV isolated from strain W-PH expressing histidine-tagged phosphatase fusion protein, was electrophoresed on a 12% SDS-polyacrylamide gel, and the phosphatase fusion protein on the gel (PufA-PhoA-his ₆ ) was isolated. At the same time, as a standard protein for quantification, histidine-tagged stilbene synthase (his ₆ -tagged stilbene synthase, STS-his ₆ , Korean Patent Application No. 10-2017-0121109) was electrophoresed at various concentrations. . Thereafter, the proteins separated from the SDS-polyacrylamide gel were transferred to a nylon membrane, and then an anti-histidine-tag antibody (MBL life science, Japan) was bound thereto. Finally, a secondary antibody (Cell Signaling Technology, Massachusetts, USA) coupled with HRP enzyme (horseradish peroxidase) was bound to detect histidine-tag fused proteins through an enhanced chemiluminescence (ECL) reaction. As a result, when detecting a phosphatase fusion protein in CMV-PH, it was detected as a single band at a position corresponding to the predicted size (Fig. 4A), indicating that PufA-PhoA was accurately bound to CMV even after the CMV purification process. means it is located. In addition, the band intensities of the standard protein and phosphatase fusion protein in the western blot were quantified using the Image J program (Schneider et al. 2012. Nat. Methods 9: 671-675) (FIG. 4B). At this time, a standard curve was constructed using the quantitative values of STS-his ₆ , and the amount of PufA-PhoA contained in CMV was calculated using this curve. Assuming that CMV contains 2481 BChl a molecules per vesicle (Kim et al. 2017. J. Microbiol. Biotechnol. 27: 2173-2179), an average of 7 PufA-PhoA per CMV is assumed. It was estimated that

[Example 5] Evaluation of structural damage and membrane orientation of CMV

In this example, structural damage and membrane orientation were analyzed by measuring phosphatase activity using CMV (CMV-P) purified from WP strain. 5 is a graph showing the phosphatase activity of CMV isolated by the method of Example 2 measured by the method specified in Example 3 above. A method for obtaining the CMV ratio of the correct structure (intact forward vesicle) using the measured phosphatase activity is specified in the above solution and in FIG. 1 . Experimentally, the phosphatase activity of CMV when treated with SDS/CHCl ₃ (reflecting the amount of total CMV) and the phosphatase activity when not treated (reflecting the amount of structurally incorrect CMV) were determined, and The ratio of intact vesicles and inside-out vesicles was calculated using the formula of 1. As a result, the ratio of CMV, which is a correct structure, was measured to be 71.1%, which means that 71.1% of intact forward vesicles and 28.9% of intact reverse vesicles and damaged vesicles were present.

Using this analysis method, it is possible to quantitatively analyze how the structural state of CMV changes with time or changes in purification and reaction conditions. However, for this, the phosphatase activity of the phosphatase fusion protein contained in CMV must be stably maintained for a long time. To confirm this, as a result of measuring the total phosphatase activity of CMV-P (activity when treated with SDS/CHCl ₃ ) for one week, it was confirmed that the phosphatase activity contained in CMV was maintained very stably for at least one week (Fig. 6).

In this example, as one of the preferred embodiments, the analysis method was used to analyze how the CMV structure changes over time under specific storage conditions (10 mM Tris-Cl, pH 8.0, 10% sucrose, 4° C.). FIG. 7 shows the phosphatase activity of CMV-P measured every day (same calculation method as in FIG. 5 ) to calculate the ratio of intact forward vesicles and followed for one week. As a result, it was confirmed that intact forward follicles corresponding to about 14% of all follicles were lost, with the rate of intact forward follicles immediately after purification being 69% and the rate after one week being 55%. This information can be used to optimize storage conditions towards reducing the loss of intact forward vesicles. The structural analysis of CMV over time performed in this example is only one preferred embodiment, and those skilled in the art will be able to apply this technology to more diverse situations.

<110> SOGANG UNIVERSITY RESEARCH AND BUSINESS DEVELOPMENT FOUNDATION <120> The evaluation method for the structure of chromatophore membrane vesicle <130> P20U10C2117 <150> KR 10-2019-0160357 <151> 2019-12-05 <160> 7 <170> KoPatentIn 3.0 <210> 1 <211> 25 <212> DNA <213> artificial sequence <220> <223> pufF primer <400> 1 aagcttcgct gattttccgc gcctg 25 <210> 2 <211> 24 <212> DNA <213> artificial sequence <220> <223> pufR primer <400> 2 ctgcagctcg gcgacggcga cgcg 24 <210> 3 <211> 24 <212> DNA <213> artificial sequence <220> <223> phoF primers <400> 3 ctgcagggcg atattactgc accc 24 <210> 4 <211> 24 <212> DNA <213> artificial sequence <220> <223> phoR primers <400> 4 tctagatttc agccccagag cggc 24 <210> 5 <211> 51 <212> DNA <213> artificial sequence <220> <223> phoR2 primer <400> 5 tctagattaa tgatgatgat gatgatgacc gcctttcagc cccagagcgg c 51 <210> 6 <211> 1491 <212> DNA <213> artificial sequence <220> <223> PufA-PhoA fusion protein <400> 6 atgagcaagt tctacaaaat ttggatgatc ttcgatcccc gtcgcgtgtt cgtggctcag 60 ggcgtgttcc tgttcctcct cgcggtgatg atccacctca tcctgctgag cacccccagc 120 tacaactggc tggaaatctc tgccgccaag tacaaccgcg tcgccgtcgc cgagctgcag 180 ggcgatatta ctgcacccgg cggtgctcgc cgtttaacgg gtgatcagac tgccgctctg 240 cgtgattctc ttagcgataa acctgcaaaa aatattattt tgctgattgg cgatgggatg 300 ggggactcgg aaattactgc cgcacgtaat tatgccgaag gtgcgggcgg cttttttaaa 360 ggtatagatg ccttaccgct taccgggcaa tacactcact atgcgctgaa taaaaaaacc 420 ggcaaaccgg actacgtcac cgactcggct gcatcagcaa ccgcctggtc aaccggtgtc 480 aaaacctata acggcgcgct gggcgtcgat attcacgaaa aagatcaccc aacgattctg 540 gaaatggcaa aagccgcagg tctggcgacc ggtaacgttt ctaccgcaga gttgcaggat 600 gccacgcccg ctgcgctggt ggcacatgtg acctcgcgca aatgctacgg tccgagcgcg 660 accagtgaaa aatgtccggg taacgctctg gaaaaaggcg gaaaaggatc gattaccgaa 720 cagctgctta acgctcgtgc cgacgttacg cttggcggcg gcgcaaaaac ctttgctgaa 780 acggcaaccg ctggtgaatg gcagggaaaa acgctgcgtg aacaggcaca ggcgcgtggt 840 tatcagttgg tgagcgatgc tgcctcactg aattcggtga cggaagcgaa tcagcaaaaa 900 cccctgcttg gcctgtttgc tgacggcaat atgccagtgc gctggctagg accgaaagca 960 acgtaccatg gcaatatcga taagcccgca gtcacctgta cgccaaatcc gcaacgtaat 1020 gacagtgtac caaccctggc gcagatgacc gacaaagcca ttgaattgtt gagtaaaaat 1080 gagaaaggct ttttcctgca agttgaaggt gcgtcaatcg ataaacagga tcatgctgcg 1140 aatccttgtg ggcaaattgg cgagacggtc gatctcgatg aagccgtaca acgggcgctg 1200 gaattcgcta aaaaggaggg taacacgctg gtcatagtca ccgctgatca cgcccacgcc 1260 agccagattg ttgcgccgga taccaaagct ccgggcctca cccaggcgct aaataccaaa 1320 gatggcgcag tgatggtgat gagttacggg aactccgaag aggattcaca agaacatacc 1380 ggcagtcagt tgcgtattgc ggcgtatggc ccgcatgccg ccaatgttgt tggactgacc 1440 gaccagaccg atctcttcta caccatgaaa gccgctctgg ggctgaaata a 1491 <210> 7 <211> 1515 <212> DNA <213> artificial sequence <220> <223> PufA-PhoA-his6 fusion protein <400> 7 atgagcaagt tctacaaaat ttggatgatc ttcgatcccc gtcgcgtgtt cgtggctcag 60 ggcgtgttcc tgttcctcct cgcggtgatg atccacctca tcctgctgag cacccccagc 120 tacaactggc tggaaatctc tgccgccaag tacaaccgcg tcgccgtcgc cgagctgcag 180 ggcgatatta ctgcacccgg cggtgctcgc cgtttaacgg gtgatcagac tgccgctctg 240 cgtgattctc ttagcgataa acctgcaaaa aatattattt tgctgattgg cgatgggatg 300 ggggactcgg aaattactgc cgcacgtaat tatgccgaag gtgcgggcgg cttttttaaa 360 ggtatagatg ccttaccgct taccgggcaa tacactcact atgcgctgaa taaaaaaacc 420 ggcaaaccgg actacgtcac cgactcggct gcatcagcaa ccgcctggtc aaccggtgtc 480 aaaacctata acggcgcgct gggcgtcgat attcacgaaa aagatcaccc aacgattctg 540 gaaatggcaa aagccgcagg tctggcgacc ggtaacgttt ctaccgcaga gttgcaggat 600 gccacgcccg ctgcgctggt ggcacatgtg acctcgcgca aatgctacgg tccgagcgcg 660 accagtgaaa aatgtccggg taacgctctg gaaaaaggcg gaaaaggatc gattaccgaa 720 cagctgctta acgctcgtgc cgacgttacg cttggcggcg gcgcaaaaac ctttgctgaa 780 acggcaaccg ctggtgaatg gcagggaaaa acgctgcgtg aacaggcaca ggcgcgtggt 840 tatcagttgg tgagcgatgc tgcctcactg aattcggtga cggaagcgaa tcagcaaaaa 900 cccctgcttg gcctgtttgc tgacggcaat atgccagtgc gctggctagg accgaaagca 960 acgtaccatg gcaatatcga taagcccgca gtcacctgta cgccaaatcc gcaacgtaat 1020 gacagtgtac caaccctggc gcagatgacc gacaaagcca ttgaattgtt gagtaaaaat 1080 gagaaaggct ttttcctgca agttgaaggt gcgtcaatcg ataaacagga tcatgctgcg 1140 aatccttgtg ggcaaattgg cgagacggtc gatctcgatg aagccgtaca acgggcgctg 1200 gaattcgcta aaaaggaggg taacacgctg gtcatagtca ccgctgatca cgcccacgcc 1260 agccagattg ttgcgccgga taccaaagct ccgggcctca cccaggcgct aaataccaaa 1320 gatggcgcag tgatggtgat gagttacggg aactccgaag aggattcaca agaacatacc 1380 ggcagtcagt tgcgtattgc ggcgtatggc ccgcatgccg ccaatgttgt tggactgacc 1440 gaccagaccg atctcttcta caccatgaaa gccgctctgg ggctgaaagg cggtcatcat 1500 catcatcatc attaa 1515

Claims (7)

A method for structural analysis of photoluminal cell membrane vesicles (CMV) comprising the following partial steps:
a) a process of preparing a gene encoding a phosphatase-fused protein by conjugating a gene of a protein constituting CMV with a phosphatase gene and a process of preparing a plasmid carrying the gene;
b) transforming a purple non-sulfur bacterium using the plasmid prepared in a) above, and purifying CMV containing a phosphatase fusion protein from the strain; and
c) A process for measuring the phosphatase activity of CMV containing a phosphatase fusion protein.
The method for analyzing the structure of CMV according to claim 1, wherein the CMV is isolated from purple non-sulfur bacteria.
The method of claim 1, wherein the phosphatase catalyzes a reaction in which a water (H ₂ O) molecule is added to a phosphoric acid monoester of the substrate to separate it into an alcohol functional group and phosphoric acid. Structural analysis method of CMV, which is an enzyme that does.
The structural analysis of CMV according to claim 1, wherein the protein to which the phosphatase is fused is a protein obtained by fusion of a membrane protein and phosphatase present on the membrane of the vesicle of the photoreceptor cell membrane in accordance with the translational frame. Way.
[Claim 5] The method for structural analysis of CMV according to claim 4, wherein all or part of the nucleotide sequence of a gene encoding each protein of the membrane protein and phosphatase can be used for fusion.
a) a plasmid containing a fusion gene in which a gene encoding a membrane protein present on the membrane of photovesicle cell membrane vesicles (CMV) and a gene encoding phosphatase are transcriptionally spliced together; or
b) A composition for structural analysis of cell membrane vesicles comprising a purple non-sulfur bacterial strain transformed with a) above.
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