JP2005168316A - Method for synthesizing protein - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for synthesizing a protein targeted on or in a membrane structure using a method for synthesizing a cell-free protein, for further detail, the method for synthesizing the protein having a physiological activity comprising adding a cellular membrane component to a cell-free protein reagent without containing an oxidizable type substance under reducing conditions. <P>SOLUTION: This method for synthesizing the protein targeted on or in the membrane structure comprises adding the cellular membrane component to the cell-free protein reagent satisfying the following conditions (a) and (b). (a) without containing a glutathione component at ≥2 mM concentration and (b) containing a reducing agent at ≥0.2 mM concentration. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a method for synthesizing a “protein targeted on or in a membrane structure” using a cell-free protein synthesis method, and more particularly, to a cell-free protein reagent under reducing conditions that does not contain an oxidized substance. The present invention relates to a method for synthesizing a protein having physiological activity, characterized by adding a sex membrane component.

  Based on recent progress in genetic engineering and molecular biology and the results of various genome sequencing, the focus of research is to express the protein of the target gene and comprehensively analyze its function and structure. It has come to come off. Among them, proteins that are particularly well-researched are proteins present in cells, and are being researched by expressing them by various methods based on gene sequences. In particular, from the viewpoint of high throughput, in recent years, it has become common to use exhaustive synthesis using a cell-free protein synthesis method.

  The cell-free protein synthesis method synthesizes proteins regardless of cell growth by preparing various cell line debris instead of living cells and adding substrates and energy sources as necessary.・ It is a technology to express. Therefore, it is possible to synthesize almost any kind of protein and its application range is very wide. Currently known cell-free protein synthesis systems include E. coli cell debris, mammalian reticulocyte extract, and plant seed extract. By using these, protein synthesis is possible in a cell-free system.

In particular, in recent years, a method using a high-efficiency wheat germ cell-free protein extract has attracted attention (for example, see Non-Patent Document 1). This method is a method in which protein synthesis ability is enhanced by reducing the contents of tritin and other ribotoxins, which are components that inhibit protein synthesis when contained in a wheat germ extract. As a method for synthesizing proteins derived from eukaryotes, the conventional method using rabbit reticulocytes has been mainly used. However, for the reason of low yield, many contaminating proteins, etc., it is necessary to analyze the function of the synthesized protein. Seems to be a less suitable way.
Proc. Natl. Acad. Sci. USA 96 (2), 559-564 (2000)

On the other hand, in this improved method using wheat germ, a protein can be obtained in a high yield by using a multilayer method together (see, for example, Non-Patent Document 2). In addition, because the extract is prepared from dormant embryos as raw materials, there are few contaminating proteins, which is advantageous when analyzing the function of the synthesized protein. Analysis of proteins from humans and other eukaryotes Has begun to be widely used.
FEBS Letter 514, 102-105 (2000)

  In addition, in cell-free protein synthesis methods, it is relatively easy to incorporate stable isotopes, selenomethionine, and the like into proteins, and there are great expectations for application to NMR and X-ray crystal analysis.

  On the other hand, research on proteins that do not exist in the cytoplasm, that is, secreted proteins, proteins localized in organelles, membrane proteins, and so-called “proteins targeted on or in membrane structures” The current situation is behind that of the internal protein. This is due to the fact that in expression using cells and the like, secretion and localization are rate limiting, and the expression is often not normal. In addition, many of them need to be subjected to various modifications after translation or embedded in a membrane, and it is often difficult to use a cell-free protein synthesis method.

In order to solve this problem, a method of fragmenting the endoplasmic reticulum that is an intracellular organ and adding the fraction to a cell-free protein synthesis system was considered. A fragment of an endoplasmic reticulum that is an intracellular component is usually called a microsomal fraction, and generally a method for preparing animal-derived microsomes is known (see, for example, Non-Patent Document 3). This microsomal fraction can also be obtained from organs such as animal pancreas, liver and fallopian tubes, and yeast cells. It is known that the prepared microsomal fraction can be coupled with a cell-free protein synthesis system derived from rabbit reticulocytes and a cell-free protein synthesis system derived from wheat germ.
Methods in Enzymology, 1983, Vol. 96, 84-93

However, with these methods, the reality is that they have been used mainly for the study of translocation of membrane proteins, and there are only a few examples of analyzing the functions such as enzyme activity of the synthesized proteins. is there. In Non-Patent Document 4, β-Hexosaminidase B is synthesized in non-patent document 5 and tissue-type plasminogen activator (t-PA) is synthesized in a case where a rabbit reticulocyte cell-free system and canine pancreatic microsomes are combined. It has been reported. (Β-Hexosaminidase B is an enzyme localized in lysosomes, and t-PA is a kind of secretory serine protease.) In these examples, activity measurement is performed. It is described in the paper that the addition of oxidized glutathione is essential to these reaction systems. However, since the cytoplasm is usually in a reduced state, it can be said that it is unnatural to perform protein synthesis under oxidizing conditions.
J. et al. Biol. Chem. 263 (26), 13463-13469 Biochem J. et al. 286, 275-280 (1992)

  Moreover, in the analysis of t-PA, only the analysis example which used the isolate | separated membrane fraction as a sample is shown, and it does not illustrate about the analysis example which used directly the reaction liquid after cell-free protein synthesis | combination. It is highly possible that the cell-free protein synthesis system using rabbit reticulocytes has a high analysis background due to the high activity level of contaminating proteins. In particular, in analysis using mammal-derived proteins, it can be said that it is preferable not to use a mammalian cell-free protein synthesis method such as rabbit reticulocytes from the viewpoint of subsequent analysis.

In that regard, wheat cell-free protein synthesis (particularly, a method using a high-efficiency wheat germ cell-free protein extract (see, for example, Non-Patent Document 6) and a membrane fraction are coupled to produce a physiological It can be said that it is preferable to synthesize “proteins targeted on or in membrane structures” that have activity, but there are currently few examples of measuring activity etc. in wheat germ cell-free protein synthesis systems. There is an urgent need to pursue and improve the cause.
Proc. Natl. Acad. Sci. USA 96 (2), 559-564 (2000))

  For these reasons, there has been a demand for a method for synthesizing a “protein targeted on or in a membrane structure” having physiological activity using a wheat cell-free protein synthesis method. That is, an object of the present invention is to add a cellular membrane component to a plant-derived cell-free protein synthesis system to synthesize secreted proteins, proteins localized in organelles, membrane proteins, etc. in a form having physiological activity. To provide a method. It is also to supply reagents and kits that achieve this, and proteins synthesized by this method.

As a result of diligent research to solve these problems, the present inventors have completed the present invention. That is, the present invention has the following configuration.
(1) A method for synthesizing a “protein targeted on or in a membrane structure” using a cell-free protein synthesis method, wherein a cell-free protein reagent that satisfies the following conditions (a) and (b): A method for synthesizing a protein having physiological activity, comprising adding a cellular membrane component.
(A) Glutathione component is not contained at a concentration of 2 mM or more (b) Reducing agent is contained at a concentration of 0.2 mM or more (2) Dithiothreitol (DTT) or 2-mercaptoethanol is used as the reducing agent The protein synthesis method according to (1).
(3) The method for synthesizing a protein according to (1), wherein the cellular membrane component is derived from an animal or a plant.
(4) The protein synthesis method according to (4), wherein the cellular membrane component is canine pancreas-derived microsome.
(5) The method for synthesizing a protein according to (1), wherein the cellular membrane composition prepared is added to a buffer solution having the following constitution (c) and / or (d).
(C) The concentration of triethanolamine contained in the buffer solution is 40 mM or less. (D) Similarly, the sucrose concentration is 0.2 M or less. (6) Prepared in a buffer solution containing (e) and / or (f). The method for synthesizing a protein according to (1), wherein the cell membrane composition thus prepared is added.
(E) Zwitterion buffer component (f) Sugar alcohols or trehalose (7) The extract used for cell-free protein synthesis is derived from plants, animals or fungi, (1) A method for synthesizing the described protein.
(8) The protein synthesis method according to (7), wherein the extract used for cell-free protein synthesis is derived from wheat germ.
(9) The method for synthesizing a protein according to (8), wherein the extract used for cell-free protein synthesis is prepared by a method with little contamination with a protein synthesis inhibitor.
(10) The protein having physiological activity according to (1), wherein the protein targeted on or in the membrane structure is a secreted protein, an organelle-localized protein, or a membrane protein Synthesis method.
(11) that the physiological activity is an enzyme activity, ligand-receptor interaction, affinity, antigen-antibody reaction, cell proliferation / secretion promotion / apoptosis induction, effect on cell morphology, effect on cell component localization The protein synthesis method according to (1), which is characterized in that
(12) A method for analyzing the function and / or structure of a protein synthesized according to the method of (1) to (11) using a reaction solution without separating a membrane fraction.
(13) A method for analyzing the function and / or structure of a protein after separating the membrane fraction after synthesizing the protein synthesized according to the method of (1) to (12).
(14) The method for analyzing the function and / or structure of the protein according to (12) or (13), wherein the protein is treated with a surfactant before analysis.
(15) Reagents and kits for use in the method according to (1) to (14).
(16) A protein synthesized according to the method described in (1) to (15).

  According to the present invention, the efficiency of the synthesis of “a protein targeted on or in a membrane structure” having physiological activity in the synthesis of cell-free protein synthesis of wheat germ other than the rabbit reticulocyte cell-free protein synthesis system has been remarkably improved. . The “protein targeted on or in the membrane structure” obtained by the method of the present invention has high activity and can use a wheat germ cell-free protein synthesis system. Thus, the crude solution can be used for activity measurement, and a simple and reliable result can be obtained as compared with the conventional method.

The present invention relates to a method for synthesizing a “protein targeted on or in a membrane structure” using a cell-free protein synthesis method, which is a cell-free protein reagent that satisfies the following conditions (a) and (b): A method for synthesizing a protein having physiological activity, characterized by adding a cellular membrane component.
(A) Does not contain glutathione component at a concentration of 2 mM or more (b) Contains a reducing agent at a concentration of 0.2 mM or more

  The term “having biological activity” as used herein means that a protein is correctly targeted on the membrane, or is subjected to post-translational modifications such as disulfide bonds and glycosylation after being targeted on or in the membrane. It means to express activity. Specifically, it indicates an activity whose activity can be measured by some method such as enzyme activity, binding assay, proliferation-inducing activity, apoptosis-inducing activity, cell shape-change-inducing activity.

  As used herein, a membrane structure specifically means a cellular membrane component, and more specifically, a membrane structure such as an endoplasmic reticulum, Golgi apparatus, peroxisome, glyoxisome, microsome, or cell membrane. Show.

  Targeting on the membrane structure here means that it is embedded in the membrane or exists on the membrane surface. Examples of the type of protein include membrane protein. Most membrane proteins have a highly hydrophobic sequence called a signal sequence at the N-terminus, are recognized by SRP (signal recognition particles) in the cytoplasm, and are transported to the endoplasmic reticulum. The carried protein invades into the endoplasmic reticulum from the N-terminus and finally becomes present on the membrane of the endoplasmic reticulum. Some types of membrane proteins exist that penetrate the membrane more than once. A membrane protein localized to the cell membrane is known to travel from the endoplasmic reticulum through the Golgi apparatus to the cell membrane. Many signal sequences are ultimately excised by the action of signal peptidases.

  Targeting into a membrane structure means that it becomes present, for example, in the endoplasmic reticulum or microsomes. Many of these proteins also have a signal sequence at the N-terminus and are recognized in the cytoplasm by SRP (signal recognition particles) and transported to the endoplasmic reticulum. Many of these proteins are subjected to disulfide bonds, sugar chain modifications, etc., then transported to the Golgi apparatus, and finally secreted out of the cell or transported to intracellular organelles such as lysosomes.

  Disulfide bond formation occurs in the protoplasmic inner layer (endoplasm) in prokaryotes such as E. coli and in the endoplasmic reticulum in eukaryotes. These interiors are characterized by being kept oxidative while the cytoplasm is in a reducing environment. Actually, disulfide bonds are formed by the action of DsbA and C in E. coli and PDF (protein disulfide isomerase) in eukaryotes. The formation of disulfide bonds is not sufficient when proteins are synthesized in an oxidative environment, and the correct disulfide bonds are formed by the comprehensive action of protein components as described above following membrane permeation. Are formed and become active.

  Protein glycosylation is a typical post-translational modification that occurs mainly in eukaryotes. First, the sugar chain is added to the endoplasmic reticulum, and then the pathway is transferred to the Golgi apparatus and converted into the mature sugar chain. The endoplasmic reticulum plays a central role in this reaction.

  As described in the section of the prior art, in the cell-free protein synthesis, microsomes, which are fragments of the endoplasmic reticulum, have been used as a field for these reactions. In particular, dog pancreas-derived myxosomes are also commercially available from Promega and others, and have been used mainly for research on translocation of membrane proteins.

  However, there are not as many examples of research on the physiological activity of synthesized proteins. Moreover, those reports are only examples of proteins synthesized under the condition that dog-derived microsomes are added to the cell-free protein synthesis system of rabbit reticulocytes and non-reducing conditions or oxidized glutathione (GSSG) is added. In addition, there are almost no cases where the physiological activity was measured using “proteins targeted on or in the membrane structure” synthesized other than the rabbit reticulocyte cell-free system.

  The glutathione component here is mainly added as oxidized glutathione (GSSG), and is used to counteract reducing properties such as DTT. In the reports so far, it is most often used at a concentration of 2 to 3 mM, and it seems that it is partially converted to reduced glutathione (GSH) by being added to a solution containing DTT. In the claims, glutathione is described as a representative substance. However, since cystine, which is a dimer of cysteine, can be used in the same manner, the absence of cystine is also included in the scope of the present invention. It is considered possible. In the present application, glutathione and cystine are collectively referred to as a glutathione component.

  In the present invention, the cell-free protein synthesis reaction solution does not contain a glutathione component at a concentration of 2 mM or more. The reason why the glutathione component is used here is that when oxidized glutathione is added to a solution containing a reducing agent, a part of the glutathione component is reduced and it is difficult to clearly define which one.

  As a method for detecting glutathione, a method using a thiol-disulfide exchange reaction with disulfide in the presence of glutathione reductase and NADPH is known.

  The reducing agent preferably contains a reducing agent selected from dithiothreitol (DTT), 2-mercaptoethanol, and the like. The concentration in the cell-free protein synthesis solution is 0.2 mM or more, preferably 0.2 to 20 mM, more preferably 1 to 10 mM, and most preferably 2 to 5 mM. Most preferably it contains DTT.

  Furthermore, the present invention is characterized by using a cellular membrane component. As the cellular membrane component, an endoplasmic reticulum component is preferably used, but the fraction centered on the endoplasmic reticulum component is preferably a fraction centered on the microsomal fraction. Here, the microsome is a fraction that can be recovered as a precipitate from a tissue homogenate by centrifugation and ultracentrifugation, and the main component is that the endoplasmic reticulum is broken by cell disruption. Microsomes are preferably derived from animal organs, but yeast-derived microsomal fractions can also be used as the endoplasmic reticulum component of the present invention. In addition to this, it is also possible to use a microbody fraction which is an organelle in a cell. It is considered that organs such as peroxisomes and glyoxisomes, which are cell-derived microbody fractions, can be used for the same purpose. The origins of microsomes that can be used in the present invention are listed as follows. Animal pancreatic-derived microsomal membrane component, animal oviduct-derived microsomal membrane component, animal liver-derived microsomal membrane component, animal-derived peroxisome, yeast-derived microsomal membrane component, plant seed-derived microsomal component, plant-derived peroxisomal membrane component, plant-derived glyoxy It is a somal membrane component. Most preferably, canine pancreas-derived microsomes are used.

  The origin of the dog is not particularly limited, but preferably, the strain is purified. A beagle dog is preferable.

  A commercially available product can be used as the canine pancreas-derived microsome, but it is preferable to use a microsome optimized for a cell-free protein synthesis system.

  Preferably, a cellular membrane fraction prepared in a buffer solution having a triethanolamine concentration of 40 mM or less and a sucrose concentration of 0.2 M or less contained in the buffer solution is preferably used. More preferably, a triethanolamine concentration is 10 mM or less and a sucrose concentration is 0.1 M or less, and most preferably a buffer solution containing no triethanolamine and sucrose is used. This is because triethanolamine and sucrose inhibit cell-free protein synthesis. Furthermore, these substances may also inhibit activities such as glycosylation of microsomes.

  Therefore, it can be said that the cellular membrane fraction used in the present invention preferably contains a zwitterion buffer component instead of triethanolamine and a sugar alcohol or trehalose component instead of sucrose.

  As the zwitterionic buffer component, Good's buffer is preferably used, and 2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid (HEPES) is more preferably used. The concentration of HEPES to be used is preferably 5 to 500 mM, more preferably 10 to 250 mM. The pH is preferably 6.8 to 8.2, more preferably 7.4 to 7.8, and most preferably 7.6. (The pH of the good buffer is preferably adjusted at 25 ° C.).

  As the sugar alcohol, sorbitol, inositol and xylitol are preferably used. Furthermore, sorbitol is preferably used. The concentration of sorbitol is preferably 0.2 to 4M, more preferably 0.3 to 2M.

The origin of the extract used for cell-free protein synthesis is not particularly limited, but is preferably derived from plants, animals, or fungi. More preferably, a method ((Proc. Natl. Acad. Sci. USA 96 (2), 559-564 (2000)) characterized in that it is derived from wheat germ and most preferably has few protein synthesis inhibitors. A cell-free protein synthesis reagent prepared according to this method is commercially available from Toyobo Co., Ltd. as a PROTIEOS ^™ Cell-free protein synthesis kit.

  Cell-free protein synthesis methods include conventional batch methods, multi-layer methods (FEBS Letter 514, 102-105 (2000)), dialysis methods (Proc. Natl. Acad. Sci. USA 96 (2), 559-564 (2000)) can be used, and the layering method and dialysis method are intermittently used in the protein reaction solution for low molecular weight substances such as ATP, GTG, and amino acids required for protein synthesis. In this method, the buffer layer containing ATP, GTG, and amino acids is layered on the reaction layer so that they are gradually diffused. The dialysis method is based on the principle that ATP, By supplying TG, amino acids, etc., and removing by-products, cell-free protein synthesis can be continued for several days. In the invention, it is preferable that the multi-layer method is suitable for functional analysis and the dialysis method is suitable for structural analysis.

  The temperature used for the cell-free protein synthesis method is not particularly limited as long as it is a temperature usually used for cell-free protein synthesis. Usually, 20-30 degreeC is performed preferably. In the method using a wheat germ extract, 20 ° C. to 26 ° C. is preferable. More preferably, 23 to 26 ° C. is used. When using an extract derived from reticulocytes or E. coli, 30 ° C. is most preferable.

  Also important in the present invention, as mentioned several times above, is cell-free protein synthesis under reducing conditions. It is known that cell-free protein synthesis is generally performed under reducing conditions, and it has been reported that cell-free protein synthesis is also performed under reducing conditions when adding cellular membrane components such as microsomes. However, almost all reports of measuring the physiological activity of the synthesized protein are cell-free protein synthesis systems using rabbit reticulocyte extract with oxidized glutathione added to the reaction system, especially using wheat germ extract There is no example in which physiological activity is measured in a protein synthesis system even in a system in which oxidized glutathione is added.

  As indicated above, wheat germ cell-free protein synthesis systems have attracted attention in recent years. Nonetheless, there is no example of adding a cellular membrane component to synthesize a “protein that is targeted on or in a membrane structure” and measuring its activity. Since the method of synthesis below was generalized, it was considered that as a result of applying it to wheat germ cell-free protein synthesis, only unexpected results were obtained. Moreover, since there has been no high-efficiency wheat germ cell-free protein synthesis system or a method for preparing cellular membrane components having higher activity in a cell-free protein synthesis solution, there is a possibility that these studies have not been made. Therefore, it can be said that the present invention has been achieved by being able to adjust all of these conditions.

  In the study in which the inventors' human tissue-type plasminogen activator (t-PA) and β-Hexosaminedase B were expressed using a wheat germ cell-free protein synthesis system, the activity was measured under reducing conditions. It was confirmed that the synthesized protein clearly has significantly higher activity (FIGS. 3 and 4). In the study using t-PA, the result is 2.5 mM DTT> 2.5 mM DTT + 2.5 M GSSG> no addition> 2 M GSSG. This result is surprisingly the opposite in the study using the cell-free protein synthesis system of rabbit reticulocyte extract, considering that the activity of these proteins synthesized under reducing conditions is almost undetectable. . However, considering that the cytoplasm is originally in a reduced state and cell-free protein synthesis should reflect that, it can be considered that this result is more natural.

  In this study, t-PA activity was successfully measured without purification of the membrane fraction. The activity measurement in such a crude system has never been reported so far, and can be considered to be caused by the property that there are few contaminating proteins in the wheat germ. This seems to be one of the analysis methods that could not have been achieved without the present invention.

  This time, we have not specifically shown the protein (membrane protein) targeted on the membrane having physiological activity, but it is possible to synthesize several types of membrane proteins using the present invention and to undergo sugar chain modification. It has been confirmed that it can be easily inferred that it has physiological activity similar to the protein shown above.

  In this series of experiments, self-prepared canine pancreas-derived microsomes were used. However, even if commercially available canine pancreas-derived microsomes were used, the efficiency was assumed to be almost the same, but the tendency was almost the same.

  The “protein targeted on or in the membrane structure” as used in the present invention refers to a secreted protein, an organelle-localized protein, a membrane protein, and the like.

  The physiological activity referred to in the present invention is not particularly limited as long as it can be measured as activity, but is not limited to enzyme activity, ligand-receptor interaction, affinity, antigen-antibody reaction, cell proliferation / secretion promotion / apoptosis induction, cell morphology This refers to the effects on cell and the effects on the localization of cellular components.

  After that, using the method of the present invention, functional analysis of secreted proteins, organelle localized proteins, membrane proteins, etc., which has been considered difficult to analyze, will be effective and high-throughput. Can be considered. Specifically, it may be widely used for applications such as receptor ligand search and drug target screening. Further, it can be applied to structural analysis.

  Examples of the present invention will be described below to further clarify the effects of the present invention. However, the scope of the present invention is not limited by these examples.

Example 1 Preparation of Buffer Solution for Cellular Membrane Composition Preparation A method for preparing a buffer solution for cell membrane composition preparation is described below.
(1) Dissolve 238 g of HEPES in sterilized water and adjust the pH to 7.5 with 1 mol / liter potassium hydroxide solution. Then make up to 1 liter with sterile water to make a 1 mol / liter solution of HEPES. At the time of use, it is adjusted to the required concentration and used.
(2) 182 g of sorbitol is weighed and dissolved in sterile water to make 500 ml. This is used as a 2 mol / liter sorbitol solution, which is prepared to the required concentration at the time of use.
Based on these solutions, the following buffer systems were prepared and used.

Example 2 Preparation of Endoplasmic Reticulum Component (Microsome Fraction) (1) Preparation solution A (50 mM HEPES, pH 7.5, 50 mM potassium acetate, 6 mM acetic acid) obtained by ice-cooling pancreas extracted from a dog (beagle dog) (Magnesium, 1 mM-ethylenediaminetetraacetic acid, 1 mM-dithiothreitol, 0.5 mM-phenylmethanesulfonyl fluoride, 0.25 M-sorbitol) and the weight was measured and then cut into pieces.
(2) The preparation solution A was added in an amount four times the weight of the pancreas and well homogenized. This solution was centrifuged at about 1,000 × g for 10 minutes, and the supernatant was collected. The supernatant was centrifuged again at approximately 10,000 × g for 10 minutes, and the supernatant was collected in the same manner.
(3) Put this supernatant solution in a centrifuge tube, and layer 1/3 volume of preparation solution B (with the same composition as preparation solution A except that the sorbitol concentration was 1.3 M) on the bottom of the centrifuge tube, Centrifuged at 140,000 xg for 2.5 hours.
(4) The precipitate is collected and suspended in the preparation liquid C (50 mM-HEPES, pH 7.5, 1 mM-dithiothreitol, 0.25M-sorbitol) in an amount equal to the weight of the pancreas in (2) above, and homogenized well. did. A small amount of this solution was diluted with 1% -SDS solution, and the OD280 value was measured. At this time, when the OD280 value was higher than 40 OD, it was diluted with the preparation solution C so as to have a 40 OD concentration. On the contrary, when it was thinner than 40 OD, ultracentrifugation was again performed by the method described in (3), and the suspension was suspended in a smaller amount of Preparation C.
(5) To this solution, an equal amount of Preparation Solution D (preparation solution C added with 50 mM EDTA) was added and allowed to stand at 4 ° C. for 15 minutes.
(6) Put this solution in a centrifuge tube, and layer 1/3 volume of preparation solution E (with the same composition as preparation solution C except that the sorbitol concentration is 0.5 M) on the bottom of the centrifuge tube. Centrifugation was performed at 000 × g for 1 hour.
(7) The precipitate was collected, suspended in half of the preparation solution C prepared in (4) above, and well homogenized. A small amount of this solution was diluted with 1% -SDS solution, and the OD280 value was measured. At this time, when the OD280 value was higher than 40 OD, it was diluted with Preparation Solution C so as to have a 40 OD concentration. On the contrary, when it was thinner than 40 OD, it was ultracentrifuged again by the method described in (6) and suspended in a smaller amount of Preparation C.
The cellular membrane composition thus prepared was used for cell-free protein synthesis.

Example 3: Cloning of human Tissue-type plasminogen activator (t-PA) and β-Hexosaminidase B gene and preparation of mRNA for cell-free protein synthesis Human β-Hexosaminidase and B Tissue-type plasminogen-activator gene (T-PA) The sequence of is already known, and primers for PCR amplification were synthesized from the database based on the sequence. For amplification of β-Hexosaminidase B, SEQ ID NOs: 1, 2 and t-PA were amplified using the primers shown in SEQ ID NOs: 3, 4 and using KOD-Plus- (manufactured by Toyobo) according to the instruction manual. . After purification of the amplified product (MagExtractor-PCR & Gel Clean Up-: used by Toyobo), the β-Hexosaminidase B amplification product was digested with SalI and the t-PA amplification product was digested with BamHI, respectively. , EcoRV-SalI and EcoRV-BamHI sites were inserted to obtain pEU-BHMB and pEU-t-PA. The plasmid was prepared by transforming E. coli JM109. Specifically, after purification using a plasmid purification kit manufactured by QIAGEN, phenol / chloroform treatment was performed and the purified product was used. The synthesis of mRNA was performed according to the instruction manual using Thermo T7 RNA polymerase (manufactured by Toyobo) using the prepared plasmid as a template. After the synthesis, gel filtration purification (sterilized water) was performed using a G-25 microspin column (manufactured by Amersham Bioscience), and then adjusted to 1 μg / μl.

Example 4 Synthesis of Human β-Hexosaminidase B and Tissue-type Plasminogen Activator (t-PA) by Cell-Free Protein Synthesis Cell-free protein synthesis was performed using the mRNA prepared in Example 3 above. For cell-free protein synthesis, a high-efficiency wheat embryo cell-free protein synthesis kit “PROTEIOS ^™ ” (manufactured by Toyobo) was used, and a synthesis reaction was performed according to the multi-layer method described in the instruction manual. This kit is described in Proc. Natl. Acad. Sci. USA 96 (2), 559-564 (2000). In this examination, a cellular membrane composition (40 OD) was added at 15% per reaction system and examined. The reaction system referred to here is a reaction solution in the lower layer of the multi-layer method. However, if the mRNA concentration (prepared in Buffer mix) described in the instruction manual is used, the volume will exceed if the method is followed. Therefore, mRNA of 1 μg / μl (prepared in sterile water) should be used. The final composition was adjusted to be the same. In this study, since it was necessary to change the concentration of the reducing agent or the like, basically the reaction buffer was Proc. Natl. Acad. Sci. USA 96 (2), 559-564 (2000), FEBS Letter 514, 102-105 (2000). According to these documents, the final concentration of the reducing agent DTT is about 2.5 mM. The experiment was carried out on the assumption that the reducing agent (final concentration of about 0.13 mM) contained in the wheat germ extract was negligible and could be ignored. Other conditions and the like were carried out in accordance with the instruction manual of the kit and the above literature. The cell-free protein synthesis reaction was performed at 26 ° C. for about 16 hours using a polystyrene 96-well plate.

Example 5: Pretreatment of enzyme activity measurement Pretreatment of a sample after cell-free protein synthesis was performed according to the following method. The pretreatment of β-Hexosaminidase B is described in J. Org. Biol. Chem. 263 (26) 13463-13469 (1988). Specifically, 10 μl of sample was dissolved in 90 μl of Buffer A (20 mM Na ₂ HPO ₄ , 12 mM citric acid, 0.1% Nonidet-P40 (pH 4.4)), incubated at 51 ° C. for 2 hours, and then 15,000 r. . p. m. For 10 minutes. Activity was measured using the supernatant as a sample.
t-PA was pretreated in the same manner after synthesis. Specifically, 10 μl of the synthesis completion solution was added to 90 μl of PBS (−) and 0.01% Tween 80, left on ice for 10 minutes or more, and the supernatant was used as a sample for activity measurement.
After the synthesis of t-PA, the membrane fraction was also purified. This operation is performed in Biochem. J 286, 275-280 (1992). Specifically, 500 μl of a solution for sucrose density gradient centrifugation (0.5 M sucrose, 100 mM KCl, 50 mM HEPES (pH 7.6)) is added to a 1.5 ml ultracentrifuge tube, and 100 μl of the reaction end solution is added thereto. After layering, 150,000 r. p. m. And centrifuged for 20 minutes. After centrifugation, the precipitate was dissolved in 75 μl of PBS (−), 0.01% Tween 80 solution and used as a sample for activity measurement.

Example 6: Activity measurement of human β-Hexosaminidase B and Tissue-type plasminogen activator (t-PA) The activity of human β-Hexosaminidase B Biol. Chem. 263 (26) 13463-13469 (1988). Specifically, 10 μl of the pretreatment solution was added to 50 μl of 1.2 mM 4-Methylumbeliferyl-N-acetyl-β-D-glucosamine hydrate (4-MU-GluNAc) (manufactured by Sigma) / Buffer A at about 37 ° C. After reacting for 1 hour, 10 times the amount of 0.17M Na ₂ CO ₃ (pH 10.0) was added, and fluorescence was measured (excitation wavelength: 355 nm, measurement wavelength: 460 nm).
The measurement of t-PA activity was performed using the ANIOPHARM ARMoSense Human tPA Chromogenic Activity Assay Kit (reference: J. Clin. Invest. 88, 1067 (1991)). The principle of measurement is that plasminogen is activated by t-PA and its protease activity is colorimetrically determined using a synthetic substrate. Specifically, Assay diluent 10 μl, Fibrinogen Fragments 10 μl, Plasgenogen 10 μl and activity measurement sample 10 μl were mixed, heated at 37 ° C. for about 30 minutes, and then added with Plasmin substrate 20 μl for about 30 minutes. The absorption at 405 nm was measured.
FIG. 1 shows the measurement results of the activity of human β-Hexosaminidase B when the reaction end solution is used as a sample. As can be understood from the figure, a strong activity was observed in a cell-free protein synthesis solution synthesized under conditions where DTT was present at a concentration of about 2.5 mM. As the reduction state became weaker, the activity tended to decrease.
FIG. 2 shows the measurement results of the activity of human t-PA when the reaction end solution is used as a sample. As can be seen from the figure, strong activity was confirmed only during synthesis under reducing conditions. In addition, no t-PA activity measurement example using a crude reaction completion solution has been reported so far, and this is presumed to have been made possible by using a wheat hyuga cell-free protein synthesis system.
FIG. 3 and FIG. 4 show an activity measurement example using a sample obtained by purifying the membrane fraction by sucrose density gradient centrifugation. In all cases, the activity of t-PA synthesized under reducing conditions is significantly high, and GSSG is added to counteract DTT used in many literatures, and proteins obtained under reducing conditions are also used with -DTT. The activity was less likely to be expressed.

  The method of the present invention enables effective and high-performance analysis of secreted proteins, organelle-localized proteins, membrane proteins, etc., which have been difficult to synthesize in the form of physiological activity in cell-free protein synthesis. Useful for increasing throughput. Specifically, it may be widely used for applications such as receptor ligand search and drug target screening. Further, it can be applied to structural analysis. Analysis of “proteins targeted on or in membrane structures” is delayed from the analysis of proteins present in the normal cytoplasm, despite the importance of drug discovery. is the current situation. In the future, it is considered that the use of this method makes it possible to easily analyze “a protein targeted on or in a membrane structure” and contribute greatly to the industry.

The figure which shows the activity measurement result of the synthetic | combination human (beta) -Hexosaminidase B. FIG. The vertical axis shows the fluorescence intensity (enzyme activity). Presence / absence of β-Hexosaminidase B mRNA and microsomal fraction in a cell-free protein synthesis reaction solution and addition of an oxidizing / reducing agent into the cell-free protein synthesis reaction solution (+ DTT: 2.5 mM DTT, −DTT: 0 mM DTT, The correlation with the physiological activity (enzyme activity) of the synthetic protein of + GSSG: 2mM GSSG is shown. (Accurately, 0 mM DTT is slightly brought in from the wheat germ extract, but was considered to be negligible because it was less than 0.2 mM) (from the left in the figure, in the order of + mRNA-Microsome, + mRNA + Microsome, -mRNA + Microsome) Displayed with The figure which shows the activity measurement result of the synthesized Tissue-type plasminogen activator (t-PA). Color development (enzyme activity) is shown on the vertical axis. Tissue-type plasminogen activator (t-PA) Presence / absence of mRNA and microsome fraction in a cell-free protein synthesis reaction solution, and addition of an oxidizing / reducing agent into the cell-free protein synthesis reaction solution (+ DTT: 2.5 mM DTT, -DTT: 0 mM DTT, + GSSG: 2 mM GSSG) shows the correlation with the physiological activity (enzyme activity) of the synthetic protein. (Accurately, 0 mM DTT is slightly brought in from the wheat germ extract, but was considered to be negligible because it was less than 0.2 mM) (from the left in the figure, in the order of + mRNA-Microsome, + mRNA + Microsome, -mRNA + Microsome) Displayed with The figure which shows the activity measurement result in the separation membrane fraction of the synthesized Tissue-type plasminogen activator (t-PA). The vertical axis shows the fluorescence intensity (enzyme activity). Tissue-type plasminogen activator (t-PA) Presence / absence of mRNA and microsome fraction in a cell-free protein synthesis reaction solution, and addition of an oxidizing / reducing agent into the cell-free protein synthesis reaction solution (+ DTT: 2.5 mM DTT, The correlation with the physiological activity (enzyme activity) of the synthetic protein of + GSSG: 2mM GSSG is shown. (Displayed in the order of + mRNA + Microsoft, -mRNA + Microsoft from the left in the figure) The figure which shows the activity measurement result in the separation membrane fraction of the synthesized Tissue-type plasminogen activator (t-PA). The vertical axis shows the fluorescence intensity (enzyme activity). Tissue-type plasminogen activator (t-PA) Presence / absence of mRNA and microsome fraction in a cell-free protein synthesis reaction solution, and addition of an oxidizing / reducing agent into the cell-free protein synthesis reaction solution (+ DTT: 2.5 mM DTT, The correlation with the physiological activity (enzyme activity) of the synthetic protein of + DTT + GSSG: 2.5 mM DTT + 2.5 mM GSSG, -DTT: 0 mM DTT) is shown. (Accurately, 0 mM DTT is slightly brought in from the wheat germ extract, but was considered to be negligible because it was less than 0.2 mM) (In the figure, displayed in the order of + mRNA + Microsome, −mRNA + Microsome from the left)

Claims (16)

A method of synthesizing a “protein targeted on or in a membrane structure” using a cell-free protein synthesis method, wherein a cell-free protein reagent that satisfies the following conditions (a) and (b) is added to a cell membrane A method for synthesizing a protein having physiological activity, which comprises adding a component.
(A) Does not contain glutathione component at a concentration of 2 mM or more (b) Contains a reducing agent at a concentration of 0.2 mM or more The method for synthesizing a protein according to claim 1, wherein dithiothreitol (DTT) or 2-mercaptoethanol is used as the reducing agent. The method for synthesizing a protein according to claim 1, wherein the cellular membrane component is derived from an animal or a plant. The method for synthesizing a protein according to claim 4, wherein the cellular membrane component is canine pancreas-derived microsome. The method for synthesizing a protein according to claim 1, wherein the cellular membrane composition prepared in a buffer solution having the following constitution (c) and / or (d) is added.
(C) Triethanolamine concentration contained in the buffer solution is 40 mM or less (d) Similarly, the sucrose concentration is 0.2 M or less. The method for synthesizing a protein according to claim 1, wherein a cellular membrane composition prepared in a buffer solution containing the following (e) and / or (f) is added.
(E) Zwitterion buffer component (f) Sugar alcohols or trehalose The method for synthesizing a protein according to claim 1, wherein the extract used for cell-free protein synthesis is derived from a plant, an animal, or a fungus. 8. The protein synthesis method according to claim 7, wherein the extract used for cell-free protein synthesis is derived from wheat germ. 9. The method for synthesizing a protein according to claim 8, wherein the extract used for cell-free protein synthesis is prepared by a method with little contamination with a protein synthesis inhibitor. The method for synthesizing a protein having physiological activity according to claim 1, wherein the protein targeted on or in the membrane structure is a secreted protein, an intracellular organelle localization protein, or a membrane protein. Physiological activity is characterized by enzyme activity, ligand-receptor interaction, affinity, antigen-antibody reaction, cell growth / secretion promotion / apoptosis induction, effects on cell morphology, cell component localization A method for synthesizing the protein according to claim 1. A method for analyzing the function and / or structure of a protein synthesized according to the method of claims 1 to 11 using a reaction solution without separating a membrane fraction. A method for analyzing the function and / or structure of a protein after separating the membrane fraction after synthesizing the protein synthesized according to the method of claims 1-12. The method for analyzing the function and / or structure of a protein according to claim 12 or 13, characterized in that the protein is treated with a surfactant before analysis. Reagents and kits for use in the method according to claim 1. A protein synthesized according to the method of claims 1-15.

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