JP2009063487A - Retrieval method of protein - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a retrieval method of functional protein. <P>SOLUTION: This method for retrieving protein-specific expression, when a material is supplied to a living body includes the processes (1) of supplying a selected material to one living thing or living thing group and a comparison material to the other same living thing or living thing group; (2) of performing the selection in both living things and groups of living things; (3) of measuring the variation in time of amount of one sort or a plurality of sorts of protein developing, after supply of the selected material and comparison material in both the living things and groups of living things; and (4) of investigating the correlation with the time between the variation of the amount of metabolic products at each time point and the variation of the amount of protein, and setting, as the candidate protein, the protein undergoing variation with time where it varies simultaneously with the amount of metabolic products, when the selected material is supplied and it does not vary simultaneously with the amount of metabolic products, when the comparison material is supplied; and (5) a process of identifying the amino acid sequence of the candidate protein. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for searching for a protein that is specifically expressed when a substance is supplied to an organism or group of organisms. This method not only makes it possible to search for proteins involved in metabolism, but also makes it possible to identify the biological or physiological function of the protein.

  Non-model organisms, in particular microbial complex systems that are difficult to cultivate, accounting for the majority of microbial communities in ecosystems, and animal and plant biological systems that contain them have been rarely used as genetic resources. However, with the recent development of molecular biological analysis means, access to these resources by the polymerase chain reaction (PCR) method and various omics analysis is gradually starting.

  Metagenomic analysis is a widely used method for obtaining genetic resources from these complex biological systems, and it exists by collecting genetic material from environmental microbial communities and clarifying its sequence. The technique of collecting all the genes to be taken is taken. However, with this method, a very large number of genes are acquired regardless of the importance of the ecosystem functions that the group is responsible for, so it is very difficult to find truly functionally important genes from them. is there.

  The present inventors have so far developed a comprehensive method for collecting genetic information only for expressed genes from such a difficult-to-culture microorganism complex system. In this method, a cDNA library is constructed without separating individual organisms from a complex microbial system, and only genes that are expressed in the library are collected. Can be collected comprehensively. For example, from a cDNA library prepared from a termite symbiotic protist population, a carbohydrate hydrolase group considered to be responsible for the main functions of the microorganism population has been successfully obtained (Non-patent Documents 1 and 2). However, even in this method, there is a difference between the obtained gene and the function of the actual microbial complex, that is, a useful biochemical reaction caused by a metabolic process or an biologically meaningful biological reaction. Correlations cannot be discussed, and it is difficult to obtain genetic information related to reactions that are actually important in industry. For this reason, in the development of useful gene resources from complex microbial populations or difficult-to-cultivate unused microorganisms, the development of a technology capable of correlating metabolic pathways and genetic information is awaited.

  In this context, Rantaleinen et al. (3) recently investigated the differences in plasma profiles between mouse models transplanted with human prostate cancer grafts and control animals, and correlated between specific metabolites and proteins. Found that there is. However, they only looked at the correlation between individual differences and did not provide a solution for searching for functional proteins and gene functions in complex systems such as complex microbial populations.

Todaka, N et al. FEMS Microbiol Ecol. 59 (3): 592-599 (2007) Inoue, T et al. Gene. 11 (349): 67-75 (2005) Rantaleinen, M et al. J. Proteome Res., 5: 2642-2652 (2006)

  Conventionally, since genome sequences of non-model organisms have not been clarified, it is possible to obtain genetic information related to reactions that are actually important in the industry for gene functions obtained from non-model organisms with various functions. Was difficult. For this reason, as described above, in the development of useful gene resources from a complex microorganism population, development of a technique capable of correlating current metabolic pathways and genetic information is awaited.

The present inventors took a termite complex biological system capable of degrading plant biomass as one example, and the amount of metabolites that fluctuates over time when termites are separately fed with cellulose and starch as feeding sources. Were examined while paying attention to specific signals of ¹ H-NMR. At the same time, the amount of protein was traced by two-dimensional electrophoresis, and as a result of correlation analysis, it was assumed that the protein that fluctuates simultaneously with the amount of the above-mentioned metabolite when cellulose is used as a feeding source is a protein involved in cellulose degradation. The amino acid sequence of this protein spot was determined by N-terminal analysis and LC / MS analysis, and the protein was determined to be a protein involved in cellulose degradation as predicted from a self-made cDNA library. In addition, it is confirmed that ¹³ C-cellulose prepared from ¹³ C plants is fed to termite complex biological system, so that ¹³ C-cellulose is decomposed by the complex biological system and metabolized to ¹³ C-acetic acid. did. In addition, the protein related to the synthesis of cuticle, which is a component of the foregut crusher used for physical lignocellulose crushing, has also been shown to specifically decrease the expression level when starch is consumed, We succeeded in observing genetic information related to the tissue physiological changes of animals according to the environment. Therefore, a new methodology for searching and confirming that protein function is equal to unused gene function information from the top down using the functional group information indicated by NMR spectrum, and diagnosing systematic physiological changes of organisms according to the environment A new method has been developed this time.

  The present invention is based on the above knowledge, but the knowledge is not limited to the termite complex biological system and can be extended to a wide range of biological systems including other non-model organisms. It makes it possible to provide new methods for finding a group of proteins or genes, or their physiological or biological functions.

Summary of the Invention The present invention is summarized as follows.
According to one aspect of the present invention, the present invention is a method for searching for a protein that is specifically expressed when a substance is supplied to an organism or a group of organisms, the following steps:
(1) supplying a selected substance to one organism or group of organisms and supplying a control substance to the other same organism or group of organisms;
(2) measuring a change in the amount of one or more metabolites derived from the selected substance and the control substance over time in both the organism or group of organisms;
(3) a step of measuring, over time, fluctuations in the amount of one or more proteins expressed after the supply of the selective substance and the control substance in both the organism or group of organisms;
(4) By investigating the temporal correlation between the change in the amount of the metabolite and the change in the amount of the protein at each time point, the control substance changes simultaneously with the amount of the metabolite when the selected substance is supplied. A protein that changes with time that does not change simultaneously with the amount of metabolites when supplying
(5) identifying the amino acid sequence of the candidate protein,
Providing the method.

  In one embodiment of the present invention, the method of the present invention may further comprise the step of creating a cDNA library from the organism or biological tissue.

  In another embodiment of the present invention, the identification of the amino acid sequence of the candidate protein in the step (5) is carried out by determining the partial sequence of the amino acid sequence of the candidate substance and then determining the sequence from the cDNA library, the known sequence or the sequence thereof. This can be done on a combination basis.

  In another embodiment of the present invention, the organism is a non-model organism.

  In another embodiment of the present invention, the organism or group of organisms is a complex microbial population, or an animal or plant or an animal group or plant group containing the complex microbial population.

  In another embodiment of the invention, the complex microbial population is an intestinal flora, a symbiotic protist population, or a plant rhizosphere microbial population.

  In another embodiment of the present invention, the organism or group of organisms has cellulolytic ability. An example of such an organism or group of organisms is a termite symbiotic protist population.

  In another embodiment, the selective substance or control substance is selected from the group consisting of natural or synthetic polymer or low molecular compounds, inorganic compounds, inorganic ions, and gases.

  In another embodiment, the method further includes the step of supplying the selective substance and / or the stable isotope of the selective substance and measuring the metabolic time of the selective substance before the step (1).

In another embodiment, after the step (5), the following steps:
(6) a step of confirming the function of the protein and the gene by introducing the gene encoding the candidate protein into a host and expressing the gene or expressing the gene in a cell-free system;
Further included.

  In another embodiment, the amount of metabolite is analyzed based on the structure of the functional group of the metabolite.

  In another embodiment, the analysis is performed by NMR.

  In another embodiment, the amount of protein is analyzed by two-dimensional electrophoresis.

In another aspect related to the above invention, the present invention is also a method for searching for a plurality of proteins that correlate with each other and function cooperatively in an organism or group of organisms, the following steps:
(1) supplying a selected substance to one organism or group of organisms and supplying a control substance to the other same organism or group of organisms;
(2) measuring the change in the amount of one or more metabolites derived from the selected substance or control substance over time,
(3) measuring the change in the amount of one or more proteins expressed after the supply of the selected substance or control substance over time;
(4) A protein and / or a specific metabolite that changes simultaneously with the amount of a specific metabolite by examining the temporal correlation between the change in the amount of the metabolite and the change in the amount of the protein at each time point Selecting a protein that does not fluctuate simultaneously with the amount of
(5) determining a partial sequence of the protein selected in step (4) and identifying the protein;
Providing the method.

Definitions As used herein, the following terms used in connection with the present invention may include the following meanings.
The term “non-model organism” refers to an organism or group of organisms other than the easily cultivated and cultivated organisms with a genetic background that have been used in conventional biological research.

  The term “substance” means a natural or synthetic substance such as a factor essential for the survival of the organism or group of organisms, or the total of substances supplied to contribute to fluctuations in the metabolic system.

  The term “selective substance” refers to the substance selected above for carrying out the method of the invention.

  The term “metabolite” refers to a substance produced by a biochemical reaction in the organism or group of organisms.

  The term “complex microbial population” means a state in which a plurality of microorganisms exist sympatrically regardless of whether they are cultured or not, and play an important function for survival.

  The term “supplying” means providing a substance that can be assimilated to an organism, including feeding, administration, application, absorption, provision, and the like.

  The method of the present invention makes it possible to search for proteins involved in metabolism utilizing the supply of substances to organisms or groups of organisms and their functions. Specifically, in an organism or group of organisms, a functional protein (s) involved in the metabolism of a specific substance in order to investigate the correlation of changes over time in metabolites and expressed proteins after the supply of the substance (eg, feeding) As well as the meaningful (physiological or biological) function of the protein (s). Even if a protein having an unknown function is obtained using a conventional method, it is not easy to elucidate the function of the protein. On the other hand, the method of the present invention analyzes a protein (group) involved in metabolism of the selected substance by selecting a substance to be supplied, and selects a functionally important protein (group) from the analysis. Provides the advantage of being able to.

The present invention will be described in further detail.
The present invention is a method for searching for a protein that is specifically expressed when a substance is supplied to an organism or group of organisms, and includes the following steps:
(1) supplying a selected substance to one organism or group of organisms and supplying a control substance to the other same organism or group of organisms;
(2) measuring the change in the amount of one or more metabolites derived from the selected substance and the control substance over time in both the organism or group of organisms;
(3) a step of measuring, over time, fluctuations in the amount of one or more proteins expressed after the supply of the selective substance and the control substance in both the organism or group of organisms;
(4) By examining the temporal correlation between the change in the amount of the metabolite and the change in the amount of the protein at each time point, the control substance changes simultaneously with the amount of the metabolite when the selected substance is supplied. A process that uses a protein that changes with time that does not change simultaneously with the amount of a metabolite when it is supplied as a candidate protein,
(5) identifying the amino acid sequence of the candidate protein,
Providing the method.

  The “organism” in the present invention is intended to include all living organisms including humans, that is, prokaryotes and eukaryotes. Prokaryotes specifically include bacteria or bacterial populations. In addition, bacterial populations include complex microbial populations symbiotic to the host, such as intestinal flora or faecal samples from animals (eg, from mammals including humans, mice and rats), symbiotic protist populations (eg, termites) Symbiotic protozoan populations of insects), plant rhizosphere microbial populations, and the like. In addition, eukaryotes include fungi such as yeast, fungi, basidiomycetes (including mushrooms), animals such as vertebrates (mammals, birds, amphibians, reptiles and fishes), invertebrates (eg, arthropods, And the like, including dicotyledonous plants, monocotyledonous plants, gymnosperms, moss plants, fern plants, algae, and the like.

  Diseases such as disease model organisms or chimeric organisms created by gene knockout or recombinant technology, etc., or so-called metabolic syndrome model organisms produced by feeding with fats and high-energy substances such as lipids This includes model organisms that are not under normal condition but are in a specific situation compared to normal.

  The present invention is also effective for non-model organisms. These organisms, as defined above, have many unknown aspects and are generally difficult to culture, such as, but not limited to, organisms such as symbiotic protist populations, gut microbiota, environmental microbial populations or the like A group of organisms can be included.

  In the examples described below, termites and protozoan populations that coexist with them were exemplified and used for searching for proteins involved in cellulose degradation. It is known that protists, including woody group-degrading bacteria, methanogenic archaea, spirochetes, and other unknown bacteria, coexist in the termite gut (Prof. Morikuma Okuma et al.・ Nucleic acid / enzyme 43: 1237-1245 (1998)). Cellulose is broken down into acetic acid by protists, and it becomes a termite energy source. Thus, organisms in the method of the present invention include organisms having cellulose decomposing ability.

  Enterobacteria are bacteria that inhabit the intestines of animals including humans. For example, in the human intestine, there are intestinal bacteria which are said to be more than 100 types and more than 3000 types. It is known that intestinal bacteria live using some of the nutrients ingested by animals and form an intestinal flora while maintaining a number balance with other types of intestinal bacteria. ing. Enterobacteria grow by fermenting nutrients ingested by animals, and at the same time, improve digestion and absorption functions, activate metabolic systems, improve immunity by degrading proteins and carbohydrates and generating metabolites. It plays a role in activation, synthesis of vitamins, activation of lipid metabolism, defense against infection, etc., and helps maintain host homeostasis.

  A plant rhizosphere microbial population is a group of soil bacteria that exist in the rhizosphere of a plant and coexist with the plant. For example, rhizobia is a soil bacterium that performs symbiotic nitrogen fixation with legumes and forms a nitrogen-circulating microbial community in the rhizosphere. It is also known that this microbial population can play a role in environmental purification of pollutants such as PCB, TPH, PAH, and heavy metals, and in the case of trees, it is known to be useful for the regeneration of forests.

  The living organism or group of organisms in the present invention includes natural ecosystems such as an ecosystem in which insects feed on terrestrial plants and an ecosystem in which fish feeds phytoplankton in the hydrosphere.

  The method of the present invention comprises a protein (or gene thereof) whose function is unknown in an organism or a group of organisms or a function thereof after supplying a specific substance (for example, feeding, administration or absorption) to the organism or a group of organisms. The quantitative variation of the metabolite and the quantitative variation of the expressed protein are monitored over time, and the correlation between the metabolite and the protein is analyzed over time and quantitative variation. The target protein is searched and identified so that the candidate protein is a protein that changes simultaneously with the amount of the metabolite when supplied, and does not change with the amount of the metabolite when the control substance is supplied. As well as revealing the physiological or biological function of the protein. Each of the steps (1) to (5) of this method will be described below.

(1) Step of supplying a selective substance or a control substance to an organism (group) The first step of the method of the present invention includes supplying a selectable substance or a control substance that can be metabolized to an organism (group). As defined above, “supplying” means providing a substance that can be assimilated to an organism (s), such as feeding, administration, application, absorption, supply, and the like.
The selection substance and the control substance may be any kind of substance as long as they can be metabolized, and are selected from the group consisting of, for example, natural or synthetic high molecular compounds or low molecular compounds, inorganic compounds, inorganic ions, and gases. The Examples of these include, but are not limited to, polysaccharides (eg, cellulose, starch, mannan, inulin, etc.), lipids (eg, unsaturated fatty acids, steroids, vitamins, carotene, etc.), proteins or peptides (eg, gluten , Collagen, tryptone, peptone, etc.), small organic molecules, such as aromatic compounds (eg, polyphenol, lignin), drug compounds, nucleic acids, inorganic compounds or inorganic ions (eg, magnesium phosphate, calcium, zinc, etc.), gases Including molecules (for example, carbon dioxide, nitrogen, etc.).

  Examples of metabolites include, for example, raffinose and xylose in the case of polysaccharides, cholesterol and triglycerides in the case of lipids, glutathione and dipeptides in the case of peptides, hippuric acid, tyrosine, In the case of gas molecules such as lignin monomer, organic compounds and derivatives thereof by carbonic acid fixation and nitrogen fixation as in plants, amino acids, ammonia / nitric acid / nitrite nitrogen compounds and derivatives thereof can be exemplified.

  The substance to be supplied can be selected based on, for example, specific bait, environmental substances, geological components, atmospheric components, medicinal components, and the like. In the case of termites as described in the examples below, wood, especially cellulose, is used as a feed, so cellulose is used as an intake source, and proteins related to its metabolism, particularly cellulolytic enzymes (ie cellulases) are searched. Can be implemented. Cellulose is decomposed into glucose and enters the TCA cycle via pyruvic acid and acetyl CoA, and is metabolized to reduce the molecular weight to acetic acid or the like (Japanese Patent Laid-Open Nos. 2006-053099 and 2006-053100).

  In the present invention, a control substance is supplied to the organism (s) together with a selection substance, where the purpose of using the control substance is to induce metabolic changes in the control organism. In other words, it is possible to easily capture the changing metabolite by changing the substance to be supplied. For this reason, the selection substance and the control substance are supplied to the organism (group) simultaneously (or successively) or separately to analyze the change of the metabolite. In this case, the selected substance may be supplied to one organism (group) and the control substance may be supplied to the other same organism (group), or both the selected substance and the control substance may be supplied to one organism (group). May be. In addition, this step includes administration or non-administration (control administration) of an agent that restricts or enhances the activity of a functional factor (s) of an organism or a group of organisms; It can be replaced by chemical and physical condition control.

  Examples of the combination of the selective substance and the control substance are not limited to the following, but are cellulose and starch, wood and cellulose, normal food and specific bioactive substance, antibiotic-containing feed and antibiotic-free feed. The physiologically active substance includes, for example, medicinal ingredients contained in pharmaceuticals, health foods, and natural products. Antibiotics include, for example, cephem, penicillin, macrolide, tetracycline and the like.

  The substance to be supplied to the organism or group of organisms is not a problem as long as it is affected by the supply, but the selected substance or control substance is 5% by weight or more, preferably 10% by weight or more, more preferably 30% by weight or more. Preferably 50% or more, particularly preferably 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, or 100% by weight. The composition may be contained in an amount.

The method of the present invention may further include a step of supplying the selective substance and / or a stable isotope of the selective substance and measuring a metabolic time of the selective substance before the step (1). This step is for estimating in advance the time required for metabolism of the substance in the organism (group). A substance labeled with a stable isotope (for example, ¹³ C, ¹⁵ N, etc.) is prepared as a stable isotope by a known method. For example, it can be produced by growing a plant with ¹³ C—CO ₂ (for example, JP 2006-053099 and JP 2006-053100 of Kikuchi et al.). This is supplied to the organism (group), and the time required for metabolism of the selected substance is estimated by tracing the metabolic dynamics, and the time width of the temporal analysis of the change of the metabolite in the subsequent process is planned.

(2) In the organism (group), a process of measuring a change in the amount of the metabolite derived from the selected substance or control substance over time. After supplying the selected substance or control substance to the organism (group), one or more The amount of change in metabolites is measured over time.
As used herein, the term “time-lapsed” refers to a period of hours, days, tens of days, weeks, months, years, etc. Means.

  The metabolite is preferably a low molecular weight substance having a molecular weight of 1,000 or less in order to facilitate analysis. However, even a high molecular substance such as a polysaccharide or a protein can be analyzed for a compound that has been reduced in molecular weight through a treatment such as hydrolysis. Metabolites vary depending on the type of selected substance or control substance. Examples of metabolites are sugars or sugar chains, organic acids such as acetic acid and lactic acid, amino acids, vitamins and the like. The organism itself or the biological tissue is collected or removed and optionally lyophilized, and the metabolites contained therein are extracted with a suitable solvent such as a buffer, or the organism itself is measured non-invasively. Here, the organism itself usually refers to a microorganism or a population of microorganisms. For example, if the organism is an insect, the organism is an insect body from which the intestinal tract has been removed, and if the organism is a vertebrate, organs, muscles, brain, bones, skin, blood vessels, lymph nodes, and other tissues are included. Including.

Metabolite analysis can be performed by selecting a signal whose intensity varies specifically in an organism by supplying a specific substance by ¹ H-NMR. The selection of a specific signal focuses on the structure of each functional group such as a methyl group or an aromatic ring. For example, it is possible to divide the range of 0.5 to ¹⁰ ppm of ¹ H chemical shift into, for example, 250, and analyze the intensity change of each signal with respect to the chemical shift value to be observed in a certain structure.

¹ H-NMR is proton nuclear magnetic resonance spectroscopy, and is particularly used for determining the structure of organic compounds. Depending on the structure and type of the functional group constituting the compound, the behavior of protons contained in the group varies depending on the magnetic field, and a difference in resonance frequency occurs. This is called chemical shift. For example, methyl group protons are shifted to the high magnetic field side (low chemical shift value), and aromatic ring protons are observed to the low magnetic field side (high chemical shift value).

The measurement solvent is water or an organic solvent containing 10 wt% or more of deuterium (D) for magnetic field locking, and includes, for example, heavy water, deuterated methanol, deuterated chloroform and the like. Although it does not specifically limit in the method of this invention, Heavy water is preferable. The sample is dissolved in a solvent, and the spectrum is measured with a ¹ H-NMR apparatus. Focusing on an appropriate signal (signal derived from a metabolite) whose intensity varies with time, the type of this signal (ie, the type of functional group) is predicted from the chemical shift value.

  Examples of other methods for analyzing metabolites include vibrational spectroscopy (measurement based on infrared light absorption), mass spectrometry (measurement based on mass difference), chromatography (molecular weight, chargeability, For example, a Fourier transform infrared spectroscopic apparatus.

(3) A step of measuring a temporal change in the amount of protein expressed after the supply of the selection substance or control substance in the organism (group) In the method of the present invention, one or a plurality of substances expressed after the supply of the selection substance or control substance Measure the amount of protein over time. Proteins are extracted from the organism itself or biological tissue at each time point, subjected to two-dimensional electrophoresis consisting of isoelectric focusing and SDS-polyacrylamide gel electrophoresis (PAGE), and serial numbers are assigned to each spot on the stained gel. Add.

  Two-dimensional electrophoresis is a technique for separating proteins two-dimensionally, and in the first dimension, the proteins are separated by isoelectric point differences by isoelectric focusing on a gel with a pH gradient, Next, in the second dimension, proteins are separated by molecular weight difference by SDS-PAGE. For staining, fluorescent staining, silver staining and the like are usually used. It is convenient to use a commercially available gel, such as Invitrogen or GE. Specific examples of the two-dimensional electrophoresis method, gel preparation, buffer and the like are described in, for example, Toda, T. and Kimura, N., Jpn J. Electroph. 41: 13-19 (1997).

  For the same number of spots, the variation is determined from the intensity of the spots. For each spot, the correlation of a specific selected substance to the control substance is determined. The correlation is visualized or analyzed by an appropriate method. For example, color is classified as blue for positive correlation, red for negative correlation, and white for no correlation, and a correlation diagram of a specific selected substance and a control substance is created for all spots. (See FIG. 3). In addition, the relationship between spots can be extracted by, for example, an appropriate cluster analysis method (for example, the neighborhood coupling method).

(4) Step of determining a candidate protein by examining the temporal correlation between the change in the amount of metabolite and the change in the amount of protein at each time point. The results obtained in the above (2) and (3) Based on the metabolic reaction of interest (ie, the difference in metabolic fluctuation due to the difference in the substance to be supplied), the time course of metabolites characteristic of ¹ H-NMR and protein expression based on two-dimensional (2D) -PAGE Extract the co-correlation of the change in quantity over time. In other words, a protein that changes over time is identified corresponding to a metabolite that changes quantitatively over time and specifically with a specific selection substance.
More specifically, a protein having a target function that changes with the amount of metabolite when a selected substance is supplied and does not change simultaneously with the amount of metabolite when a control substance is supplied. It can be a protein.

  In the examples of termite cellulose feeding or starch feeding described in the examples below, from the correlation diagram shown in FIG. 3, four sets showing a positive correlation in cellulose feeding and a negative correlation in starch feeding are shown. Extracted (displayed in a box). In particular, among the protein spot groups, a spot group that has a large amount of protein expression and can be quantified stably over time is selected. As a result, cellulase was found at spot 44, while actin, a housekeeping protein as a control, was found at 109th. As this example shows, the method of the present invention provides an effective technique for finding expressed proteins that fluctuate simultaneously with changes in metabolites after the supply of a selective substance.

(5) Step of identifying the partial sequence of the protein selected in step (4) After isolating the partial amino acid sequence of the protein selected in the above (4) from the electrophoresis gel, amino acid analysis, LC / Determined by techniques such as MS analysis. For example, after the isolated protein is cleaved with a protease such as trypsin, the peptide fragments can be fractionated by high performance liquid chromatography using a gel filtration HPLC column, dialyzed and lyophilized, and then subjected to amino acid analysis. .

  Based on the determined amino acid sequence, proteins can be identified using known algorithms such as BLAST, FASTA, etc. (Toshihisa Takagi and Jun Kaneko, GenomeNet database usage (2nd edition), 1998 , Kyoritsu Publishing). Alternatively, using a probe prepared on the basis of the amino acid sequence, DNA encoding the target protein is lifted from a cDNA library prepared from a living organism or biological tissue, the nucleotide sequence is determined, and a known algorithm is similarly used. Thus, the corresponding gene and protein can be identified.

Preparation of a cDNA library from a living organism or biological tissue can be performed as follows.
MRNA is obtained from an organism or biological tissue, such as a complex microbial population, a symbiotic protist population, or the like. mRNA can be obtained by conventional techniques including extracting the total RNA from the destroyed organism or biological tissue using reagents such as guanidine thiocyanate, phenol / chloroform, and purifying mRNA using, for example, an oligo dT cellulose column. Obtainable. For extraction and purification of mRNA, it is convenient to use a commercially available kit (for example, GE, Roche, Invitrogen, Takara Bio, etc.).

  Next, a cDNA library is prepared from the mRNA obtained by the above method by a conventional method. That is, in the presence of reverse transcriptase and dNTP, cDNA synthesis is performed using oligo dT primer or random primer and mRNA as a template. Furthermore, after cleaving the RNA strand with RNase, the second strand cDNA is synthesized using the first strand cDNA as a template, the hairpin loop portion is excised using an enzyme such as S1 nuclease, and the double strand cDNA. And For further amplification, the cDNA is inserted into a phage vector, infected with E. coli, and the cDNA is subcloned. Kits (for example, Invitrogen, Stratagene, Cosmo Bio, etc.) for carrying out this series of techniques are commercially available, and a cDNA library can be prepared by using them.

  In the present invention, when targeting non-model organisms, particularly organisms such as microbial complex systems or symbiotic protists that occupy most of the microbial community in the ecosystem, multiple types of microorganisms or protists are mixed. Therefore, genetic information is very complicated. A cDNA library prepared from such a non-model organism is also called an environmental cDNA library. The method of the present invention has the advantage that it can be used effectively even in non-model organisms as described above.

  The prepared cDNA library should contain DNA encoding the protein of interest, and this library can be used for searching and identifying the protein of interest.

  In the method of the present invention, after the step (5), the gene encoding the candidate protein is introduced into a host and expressed, or expressed in a cell-free system, and the function of the target protein or the gene is confirmed. Steps may be included.

  After the amino acid sequence of the target protein is identified, DNA encoding the target protein is amplified from the above cDNA library using, for example, polymerase chain reaction (PCR), and then inserted into an appropriate vector. Then, a host cell can be transformed with this vector to express the DNA. Thereafter, for example, the expression product protein may remain unpurified, and the change in the metabolite in the cell-free reaction solution may be traced, or the target protein may be recovered and added to the appropriate cell extract to As in the case of investigating in (4), changes in metabolite groups may be traced. Furthermore, in the same manner as in general methods, the biochemical activity (for example, enzyme activity, proliferation activity, receptor-ligand activity, signal transduction activity, transcription factor activity, etc.) of the collected target protein is confirmed and a specific substance is identified. The function of the protein or its gene can be confirmed from the known information of the metabolic system. Alternatively, by transforming plant cells, animal germ cells or embryonic stem cells with the vector, a chimeric organism individual is prepared, and the biological function of the DNA is determined by observing the influence of the overexpression of the DNA. Can be confirmed.

  Introduction of foreign DNA or a foreign gene into animal cells can be performed by homologous recombination. Vectors such as plasmids, viruses (adenoviruses, adeno-associated viruses, retroviruses, etc.), artificial chromosome vectors (YAC, HAC, etc.), and promoters (eg tissue-specific promoters, event-specific promoters, viral promoters, etc.) as necessary The foreign DNA or foreign gene can be integrated into the animal cell and then introduced into the animal cell. The foreign DNA or foreign gene is usually flanked with a base sequence of about 2 to 6 kb homologous to the base sequence at the position to be inserted on the animal genome so as to sandwich the foreign DNA or foreign gene. Foreign DNA or a foreign gene can be introduced into. A selection marker for confirming that homologous recombination has occurred can be inserted into the vector. Examples of selectable markers include drug resistance genes (eg, neomycin resistance gene, kanamycin resistance gene, hygromycin resistance gene, etc.). Selectable markers can optionally include positive and negative markers. An example of a positive marker is a drug resistance gene as exemplified above, and an example of a negative marker is a thymidine kinase gene such as HSV-TK.

  Oocytes or embryonic stem (ES) cells can also be used as animal cells. In this case, foreign DNA or a foreign gene can be introduced into a cell by a method such as microinjection or microcell fusion (Japanese Patent Laid-Open No. 11-31576). Oocytes are transplanted into the uterus of temporary parent non-human animals (eg, rodents such as mice, ungulates such as cows, pigs, and sheep), and chimeric animals are produced by childbirth. After mating, select homozygous progeny. Alternatively, ES cells are introduced into blastocysts derived from non-human animals (such as mice) to generate chimeric animals, and similarly homozygous offspring are produced.

  To produce a transgenic plant from plant cells, a known Agrobacterium method can be used. Usually, foreign DNA is incorporated into a Ti plasmid and introduced into an Agrobacterium strain such as Agrobacterium tumefaciens to infect a plant. The plasmid can contain a promoter such as a cauliflower mosaic virus (CaMV) promoter and a nopaline synthase (NOS) promoter, a drug resistance gene such as a kanamycin resistance gene, and the like.

  For the transformation method, a well-known method such as virus infection, transfection, protoplast method, microcell fusion method, gene bombardment, calcium phosphate method, microinjection, etc. can be used (for example, Sambrook et al., Molecular Cloning A Laboratory Manual, Cold Spring Horbor Laboratory Press, 1989; translated by Michio Matsuhashi et al., Molecular biology of Watson Recombinant DNA, 2nd edition, 1993, Maruzen, etc.).

  Alternatively, the protein identified by the method of the present invention may be expressed in a cell-free system. As the cell-free expression system, for example, a wheat germ cell-free expression system or an E. coli cell-free expression system can be used. In particular, wheat germ cell-free expression systems are commercially available from CellFree Science, Roche Applied Science, Qiagen, Invitrogen, and the like. The cell-free expression system includes energy sources such as protein synthesis factors and ATP, preferably excludes suicide factors such as tritin, and includes amino acids and genetic information (mRNA) (for example, Ehime University Venture Business) Laboratory cell-free protein synthesizer).

The present invention further relates to a method for searching for a plurality of proteins that correlate with each other and function cooperatively in an organism or group of organisms, the following steps:
(1) supplying a selected substance to one organism or group of organisms and supplying a control substance to the other same organism or group of organisms;
(2) measuring the change in the amount of one or more metabolites derived from the selected substance or control substance over time,
(3) measuring the change in the amount of one or more proteins expressed after the supply of the selected substance or control substance over time;
(4) A protein and / or a specific metabolite that changes simultaneously with the amount of a specific metabolite by examining the temporal correlation between the change in the amount of the metabolite and the change in the amount of the protein at each time point Selecting a protein that does not vary simultaneously with the amount of (5), and (5) determining a partial sequence of the protein selected in step (4) and identifying the protein,
Providing the method.

  This method comprises the same steps as the above-described method of the present invention, but based on the finding that it can also be used to search for and identify a group of proteins that function in a cooperative manner in a metabolic process of substances. ing. For each step, what is specifically described above can be applied here.

The present invention further relates to a method for searching a function of a protein of a living organism or a gene encoding the same, and the following steps:
(1) supplying a selective substance and / or a control substance to the organism;
(2) measuring a change in the amount of one or more metabolites derived from the selected substance or control substance over time in the organism,
(3) measuring a change in the amount of one or more proteins expressed after the supply of the selected substance or control substance over time in the organism,
(4) examining a temporal correlation between the change in the amount of the metabolite and the change in the amount of the protein at each time point, and selecting a candidate protein that changes simultaneously with the amount of the specific metabolite;
(5) determining a partial sequence of the protein selected in step (4), and identifying the candidate protein as necessary;
(6) confirming the function of the candidate protein or gene thereof,
Providing the method.
For each step, what is specifically described above can be applied here.

  Conventionally, regarding gene functions or protein functions obtained from non-model organisms having a variety of functions, it has been difficult to obtain genetic information related to reactions that are actually important in the industry, but the method of the present invention. Is a protein that is a gene product using functions of non-model organisms, that is, useful biochemical reactions or biologically meaningful biological reactions that are caused by metabolic processes, using metabolite signal fluctuations as functional information. Since the strategy of tracking the co-correlation with the quantity is taken, the acquisition of gene or protein function information can be quickly reached. When the gene function is examined reductively as a protein function, an enzyme reaction is analyzed in an in vitro system, but the point that the confirmation method can be performed by NMR analysis of an actual biological system is also a point that is greatly different from the conventional method.

  At the same time, the method of the present invention succeeded in diagnosing the systemic variation of termites due to different feeding conditions at the gene expression level. From this, this method is a new technique capable of diagnosing various tissue physiological changes according to the environment that have been difficult to analyze from the two aspects of gene expression related to metabolites. Provide a means. This can also be applied to the identification and diagnosis of causes in lifestyle-related diseases caused by a combination of various environmental factors that are currently becoming problems.

  In the following examples, termite complex biological systems are taken as examples to further illustrate the present invention, but the scope of the present invention is not limited by these specific examples.

1) Prediction of cellulose metabolism time in termite complex biological system
^The plant was fixed with carbon by photosynthesis in a chamber filled with ¹³ CO ₂ and ¹³ C was incorporated. ^{The 13} C plant was heated with a 1% NaOH aqueous solution at 121 ° C. for 15 minutes, strained with gauze, and rinsed with running water. Thereafter, it was immersed overnight in a 0.6% aqueous sodium hypochlorite solution and washed overnight with running water. Drying was performed to obtain ¹³ C cellulose. Termites were raised using this as a bait, and the termite parasites from which symbiotic protists and intestinal tracts were removed over time were sampled and freeze-dried. After crushing the freeze-dried sample, 100 mM potassium phosphate buffer was added to extract a metabolite, and HSQC (Hetero-nuclear Single Quantum Coherence) measurement was performed using NMR. With the cellulose-degrading enzyme possessed by termites and symbiotic organisms, the time-lapse ¹³ C signal intensity associated with the degradation of ¹³ C-cellulose was observed to confirm the metabolic time of cellulose.

2) Isolation of mRNA and construction of cDNA library Yamato termites, termites, licorice termites, long-tailed termites, and cockroaches intestinal tracts were extracted and Solution U ™ (Trager 1934, Biological Bulletin, Vol66: 182-190) Crushed in and filtered through a 100 micron nylon mesh. The resulting suspension was centrifuged gently at 100 × g for 3 minutes to obtain a protozoan fraction. Further, it was washed 3 times with Solution U to obtain a protist fraction for RNA extraction. Intestinal tract and salivary glands were removed from termites. From the obtained intestinal tract and salivary gland, the salivary gland, foregut, midgut, and hindgut were cut out with a scalpel and washed with Solution U three times to obtain digested tissue samples for mRNA extraction. At the same time, a termite worm body from which the intestinal tract was removed was obtained. mRNA was isolated according to the instructions using Oligo-dT30 <Super> (Roche Japan), and mRNA was obtained from protists and termite tissues derived from each host termite.

  A cDNA library was constructed using 2-3 micrograms of mRNA prepared from each termite symbiotic protist group and termite tissue. Library construction was carried out by Carnici et al. For Yamato termites (Carninci, P & Hayashizaki, Y 1999, Methods Enzymol 303: 19-44), and other termite-derived ones by Kanno et al. (Maruyama, K. and S. Sugano 1994, Gene 138 (1-2): 171-174). The Yamato termite cDNA library was subcloned into a plasmid according to the instructions using SOLR strains E. coli (Novagen) and ExAssist helper phage (Novagen).

3) Tracking the amount of metabolites that change over time when two types of feeding sources are cellulose and starch. Termites were bred using cellulose and starch as feeding sources. Termite parasites from which the contents of the hindgut and the intestinal tract were removed were sampled on a regular basis and lyophilized. After crushing the freeze-dried sample, 100 mM potassium phosphate buffer was added to extract the metabolite, and ¹ H-NMR measurement was performed using a 700 MHz-NMR apparatus (FIG. 1).

4) Tracking of protein mass by two-dimensional electrophoresis 4-1) Preparation of protein extract Using 3-5 milligrams of the lyophilized sample of 2) above, protein was detected with Lysis buffer (8M Urea, 2% CHAPS, DTT 40 mM). Extracted. The protein extract was purified using 2D Clean-Up Kit ™ (GE) according to the instructions.

4-2) Two-dimensional electrophoresis of protein A purified protein extract (60 micrograms) is subjected to isoelectric focusing with Immobiline Drystripping pH 4-7 (GE) (after swelling 14 h, 1 h at 500 V, 1 h at 1000 V, 2 h at 8000 V) )did. After the electrophoresis, second-dimensional electrophoresis was performed on a 12.5% SDS-PAGE gel. The gel was stained with SYPRO Ruby protein gel stain (invitrogen) according to the instructions and detected with Typhoon (GE health science). The intensity of the protein spot was determined from the detected image using image analysis software Image J (NIH) (FIGS. 1 and 2).

5) Time-series analysis of proteins that fluctuate simultaneously with the amount of metabolite Of the ¹ H-NMR spectrum obtained in 3) above, attention was paid to a strong signal in the anomeric region (4.8-5.6 ppm) of sugar (FIG. 2). ). This is because polysaccharides, especially cellulose, occupy an overwhelming biomass as an energy source for termites, and low molecular weight saccharides will change greatly with the increase / decrease of cellulase expression. In particular, if the feeding source is cellulose and starch, glucose is the low-molecular sugar that is ultimately decomposed. However, the expression of cellulase and its cofactors is important for the degradation of macromolecular cellulose. We thought that it would change to reflect the difference in the three-dimensional structure. Therefore, the correlation coefficient between the time course of the metabolite in the sugar region of the ¹ H-NMR spectrum (upper panel in FIG. 2) and the time course of the protein obtained in 4) above, and the cellulose feeding and starch feeding The correlation coefficient in the time course of protein (lower panel in FIG. 2) was determined by Program Excel. In this case, since the sampling is within 10 points as the variation over time, the correlation coefficient for y = x was simply obtained. However, by increasing the sampling points of the variation over time, the function of n power or the surrounding Fitting to a sine function taking into account various biorhythm fluctuations becomes possible. In this correlation coefficient diagram, 2D-PAGE protein spots having a negative correlation coefficient were selected on the assumption that the expression of cellulase and its cofactors in cellulose feeding / starch feeding was reversed. Subsequently, the correlation coefficient between the intensity of the protein spot and the intensity of the sugar chain signal is changed to four 2D-PAGE spots (spots 21, 44, 65, and 109 in FIG. 2) that are remarkably different between cellulose feeding and starch feeding. Narrow down and try to determine the next protein sequence. At the time of narrowing down a series of candidates, only 2D-PAGE spots that were accurately observed over the entire time series were used in order to prevent errors during spot intensity reading.

6) Determination of protein sequence The amino acid sequence of the protein spot obtained in 5) above was determined. The electrophoresed gel was transferred to a PVDF membrane using a conventional method, and then stained with 10% acetic acid, 2.5% methanol, and 0.1% Coomassie brilliant blue solution. The stained membrane was decolorized with methanol to cut out each spot, and the amino terminal sequence was determined using the Edman degradation method. If a long sequence could not be obtained, the gel was fixed with a fixing solution (7.5% acetic acid, 5% methanol) for 15 minutes, and then 10% acetic acid, 2.5% methanol, 0.1% Coomassie brilliant. Stained with blue solution. The gel was decolorized with a fixing solution, and then a spot was cut out. The excised spot was replaced with a protease treatment buffer, digested in gel, separated by HPLC, the length of the obtained fragment was determined by mass spectrometry, the sequence was determined by Edman degradation, and the estimated mass and In addition, the intramolecular sequence was determined. The obtained sequence was subjected to blast analysis between the known known sequence and the constructed library sequence, and the functional gene related to the target condition in 5) above was identified (FIG. 3). As a result of LC / MS, it was found that spot 21 was a putative cuticle protein, spot 44 was cellulase, spot 65 was Arg-kinase, and spot 109 was actin.

According to the present invention, search for functional protein and gene function in an organism can be performed on a commercial basis. All biological species are the targets, but as a large-scale one, the search for degrading enzymes of plant biomass described in the above-mentioned examples can be considered. In recent years, plant biomass has been regarded as important as a natural resource to obtain petroleum substitute materials and energy while reducing the problem of CO ₂ credit. However, since there are no good degrading enzymes and cofactors that break down cellulose, which is a non-edible part, lowering the price has become an urgent issue worldwide. If an efficient gene search can be performed with this method, experiments on mass expression of the target protein using this gene can be quickly started, and it can be applied to huge industries such as the energy industry, transportation machinery, and chemical materials. It will have a strong impact. In addition, since the present invention can be applied to the identification and diagnosis of causes in lifestyle-related diseases caused by a combination of various environmental factors, the intestinal bacterial flora when a human ingests a plant fiber diet If it is possible to improve the autoimmunity associated with the growth of useful intestinal bacteria and to search for unknown genes related to these, it is possible to impact the food and beverage industry, the pharmaceutical industry and the like.

In termites were fed cellulose (C) and starch ^(S), shows a model of temporal metabolic variations and 2D-PAGE of time course protein variations in 1 H-NMR. Gel photographs showing 2D-PAGE protein variation over time (spots 21, 44, 65 and 109) in termites fed with cellulose (C) and starch (S), and ¹ H- of the sugar region of the metabolite NMR spectrum. The correlation analysis example of a metabolite and protein in the termite which ingested cellulose (C) and starch (S) is shown. In addition, a, b, c, and d of cellulose (C) and starch (S) in the figure represent chemical shift values (ppm) of the NMR signal, which are 5.62, 5.46.5.42, 5 respectively. .22 ppm.

Claims (13)

A method for searching for a protein that is specifically expressed when a substance is supplied to an organism or group of organisms, comprising the following steps:
(1) supplying a selected substance to one organism or group of organisms and supplying a control substance to the other same organism or group of organisms;
(2) measuring a change in the amount of one or more metabolites derived from the selected substance and the control substance over time in both the organism or group of organisms;
(3) a step of measuring, over time, fluctuations in the amount of one or more proteins expressed after the supply of the selective substance and the control substance in both the organism or group of organisms;
(4) By examining the temporal correlation between the change in the amount of the metabolite and the change in the amount of the protein at each time point, the control substance changes simultaneously with the amount of the metabolite when the selected substance is supplied. A process that uses a protein that changes with time that does not change simultaneously with the amount of a metabolite when it is supplied as a candidate protein,
(5) identifying the amino acid sequence of the candidate protein,
Including said method.   The identification of the amino acid sequence of the candidate protein in the step (5) is performed based on a sequence from the cDNA library, a known sequence or a combination thereof after determining a partial sequence of the amino acid sequence of the candidate substance. The method described.   The method according to claim 1 or 2, further comprising the step of preparing a cDNA library from an organism or biological tissue.   The method according to any one of claims 1 to 3, wherein the organism or group of organisms is a complex microorganism population, or an animal or plant containing the complex microorganism population, or an animal group or plant group.   5. The method of claim 4, wherein the complex microbial population is an intestinal flora, a symbiotic protist population, or a plant rhizosphere microbial population.   6. The method of claim 5, wherein the symbiotic protist population is a termite symbiotic protist population.   The method according to any one of claims 1 to 6, wherein the organism or group of organisms has cellulose resolving power.   The method according to any one of claims 1 to 7, wherein the selective substance or the reference substance is selected from the group consisting of a natural or synthetic polymer or low molecular compound, an inorganic compound, an inorganic ion, and a gas.   9. The method according to claim 1, further comprising a step of supplying the selective substance and / or a stable isotope of the selective substance and measuring a metabolic time of the selective substance before the step (1). the method of. After step (5), the following steps:
(6) a step of confirming the function of the protein and the gene by introducing the gene encoding the candidate protein into a host and expressing the gene or expressing the gene in a cell-free system;
The method according to claim 1, further comprising:   The method according to any one of claims 1 to 10, wherein the amount of the metabolite is analyzed based on the structure of the functional group of the metabolite.   The method according to claim 11, wherein the analysis is performed by NMR method.   The method according to any one of claims 1 to 12, wherein the amount of protein is analyzed by two-dimensional electrophoresis.

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