KR20190081732A - Method for Isolating Seven-Transmembrane Proteins from Olfactory Sensilla of Drosophila melanogaster - Google Patents

Abstract

Membrane penetration protein from the olfactory tissue of a fruit fly, such as a sensory system, using a surfactant and iron beads.

Description

[0001] The present invention relates to a method for separating 7-membrane perforation protein from olfactory tissue of Drosophila,

The present invention relates to a method for separating 7-membrane penetration protein from the olfactory tissue of fruit fly.

Drosophila In odorant melanogaster , odorant receptors (ORs) are present in the dendritic membranes of olfactory sensory neurons (OSN). The OSN is located in the third segment of the antennae and in the maxillary palps. These olfactory organs are covered with olfactory sensilla, and most olfactory senses include OSN dendrites immersed in sensory lymphatics. The olfactory senses generally have two OSNs, each OSN expressing odor-specific olfactory receptors and the olfactory co-receptor Orco (odorant receptors co-rhetor). The activity of different OSNs can be distinguished by the characteristic amplitude of OSN using single sensulum recordings (SSR) and sensitivity to each odor.

At the OSN level, extensive studies have been conducted on the peripheral olfactory system using a single ciliate record (SSR). However, studies of protein levels of insect olfactory receptors have been conducted at a basic level . In particular, there is little known whether there is any interaction between OR and intracellular proteins.

First, the olfactory receptors of the insects belong to an exceptional protein among receptors with seven membrane-penetrating domains, or olfactory receptors. The olfactory receptors of insects were initially expected to be part of GPCRs (G protein-coupled receptors), but exhibit features that are dependent on co-receptors, with a loss of sequence homology with other sensory receptor families. In addition, unlike other GPCRs, the olfactory receptors of insects exhibit similar properties to ionotropic receptors and exhibit a specific three-dimensional structure. In other words, the study of protein interaction of OR is at the basic stage because there are limitations on the experiments that can be conducted with reference to 7-membrane perforating receptors already studied.

Second, the OR of the insect does not normally migrate from the heterologous expression system to the plasma membrane, because the heterotypic expression system insufficiently imitates the inherent biological environment of the insect OSN. Because of the limited use of heterologous expression systems in the novel finding of OR-interacting proteins, studies of protein interactions in ORs have been constrained to use actual Drosophila tissue.

Third, the study of protein levels of OR requires the use of actual fruit fly tissue, but OR is expressed in small amounts in some cells, and is physically hidden in thin, rough cilia. Research on OR using such organizations has also been limited because of the difficulty in collecting and manipulating large volumes of OR-containing organizations.

The olfactory cavity receptor (Orco) is a member of the insect olfactory receptor family, and unlike other ORs, it does not respond to natural odors, but functions as a ubiquitous co-receptor of other ORs and plays an important role in the normal action of ORs . Without Orco, OR can not migrate into cells and the response of OSN to odors is suppressed. Since Orco is expressed in almost all OSNs, a study of Orco's interactome suggests an overall mechanism of action for insect olfactory as well as a methodological progression to isolate 7-mer protein in the olfactory cells of insects .

The inventors of the present invention have studied a platform for identifying unknown ORco interacting proteins. As a result, they have found a method for efficiently isolating 7-membrane penetrating protein from olfactory olfactory tissue and completed the present invention.

It is an object of the present invention to provide a method for producing a pharyngeal olfactory tissue, Crushing the separated olfactory tissue; And a step of adding a buffer to the crushed olfactory tissue and heating, to thereby provide a method for separating the 7-membrane penetrating protein from the olfactory olfactory tissue.

According to an aspect of the present invention, Crushing the separated olfactory tissue; And a step of adding a buffer to the crushed olfactory tissue and heating, thereby providing a method for separating the 7-membrane penetrating protein from the olfactory olfactory tissue.

In one embodiment of the present invention, the olfactory tissue of the fruit fly includes olfactory sensilla, odorant receptors (OR) and olfactory cortical receptors Orco (odorant receptors co-rrescor, Orco).

The step of separating the olfactory tissue can be carried out using a micro-tissue separating device devised by the inventor. Wherein the microstructure separation apparatus comprises: a tissue storage unit for storing an insect tissue to be separated; A tissue separation unit connected to one side of the tissue storage unit and having a filter for separating the microstructure; And a microstructure receiving portion connected to one side of the tissue separating portion and accommodating the separated insect microstructure. The filter of the tissue storage part may have an opening of 50 to 300 mu m in size, and the size of the opening may be adjusted according to the size of tissue to be separated.

In one embodiment of the present invention, the step of crushing the olfactory tissue may use iron beads, and the olfactory tissue may be uniformly crushed by mixing the separated olfactory tissue with iron beads and then vortexing.

The buffer may contain a surfactant for the solubilization of proteins, and the surfactant may be selected from Triton X-100, Nonidet P-40, Octyl-bD-glucopyranoside, n-Dodecyl b-Dmaltoside (DDM) and Zwittergent 3- 16 < / RTI >

In one embodiment of the present invention, the heating step may be performed at 40 to 60 ° C, and the heating time may be 10 to 60 minutes.

Using the method according to one embodiment of the present invention, it is possible to easily isolate the 7-membrane penetration protein from the olfactory olfactory tissue such as the sense cap.

Fig. 1 shows the detailed structure of the olfactory olfactory tissue.
FIG. 2 shows a process of separating total protein including a microstructure including a olfactory tissue and 7-membrane perforation protein in a fruit fly using the apparatus for separating micro-organisms of a fruit according to an embodiment of the present invention.
FIG. 3 is a graph showing the results of 7-membrane perforating protein isolated from collected micro-tissues by collecting micro-tissues including the olfactory olfactory tissue using the apparatus for separating micro-organisms from flour according to an embodiment of the present invention.
FIG. 4 is a graph showing the degree of solubilization of 7-membrane peroxidation protein of Drosophila olfactory membrane according to the type of surfactant and the degree of solubilization of 7- Membrane through protein separation.

Hereinafter, one or more embodiments will be described in more detail by way of examples. However, these embodiments are intended to illustrate one or more embodiments, and the scope of the present invention is not limited to these embodiments.

Experimental Method

1. Drosophila

Drosophila used in the experiment was obtained from Bloomington Drosophila Stock Center (NIH P40OD018537), w ; Orco-GAL4, UAS-EGFP :: Orco; Orco ¹ was provided by Sion Lee.

All Orco experiments in which EGFP was labeled showed w ; Orco-GAL4, UAS-EGFP :: Orco; Was carried out in ^{Orco 1 (Orco-GAL4 (Wang} et al, 2003);. UAS-EGFP :: Orco (Benton et al, 2006);. Orco 1 (Larsson et al, 2004).). Genetic background control experiments were performed using w ; Orco-GAL4, UAS-myr :: GFP (myr :: GFP (Pfeiffer et al., 2010)); Orco ¹ .

2. Tissue Isolation and Protein Isolation

For the head lysate, 100 Drosophila heads (50 females, 50 males) with intact form antennas were placed in tubes and dissected on dry ice.

Drosophila microstructure separation apparatus designed by the inventor was used for the production of tissue flies. The Drosophila microstructure separator is composed of two 50 ml tubes connected by two hollow lids and a sieve having a metal mesh (pore size 180 占 퐉 ² ) It is located between the lids.

The use of the Drosophila microstructure separator was as follows. The Drosophila microstructure separator was assembled into a complete state and stored at -80 ° C for at least 30 minutes and frozen. Then, to separate the fruit fly tissue by size, the fruit fly separator was actively vortexed, and the sieved microstructure was transferred to a new tube in this process. Shortly afterwards, three steel beads (3 mm diameter) were added to the tube containing the microstructure and vortexed for 2 minutes to homogeneously grind the Drosophila microstructure.

To the pulverized Drosophila microstructure immediately, 300 μl of dissolution buffer was added and homogenized by vortexing for 1 minute. The mixture was then centrifuged at 4 [deg.] C (Zwittergent 3-16 or 10 [deg.] C using SDS) and 10,000xg for 5 minutes to remove the precipitate. The supernatant of the intermediate stage was transferred to a new tube, and further centrifuged to obtain a solution of microfollicular microfollicles containing protein (hereinafter referred to as tissue lysate).

Lysis buffer was prepared by adding cOmplete ™ EDTA-free Protease Inhibitor Cocktail (Roche, 04693159001) to a dilution buffer (GFP-Trap®_A protocol; ChromoTek, Germany) containing 1% (v / v or w / v) Solution. (Bioseang, T1027), Triton X-100 (Bio Basic, TB0198), Nonidet P-40 (Bio Basic, NDB0385), Octyl-bD-glucopyranoside (Fluka, 75083), n-Dodecyl b- Dmaltoside (DDM; ThermoFisher, 89903), CHAPS (Sigma-Aldrich, C5070), CHAPSO (Calbiochem, 220201) and Zwittergent 3-16 (Calbiochem, 693023).

Protein quantification was performed using the Pierce ™ BCA Protein Assay Kit (ThermoFisher, 23227) and the final tissue lysate was stored at -80 ° C.

FIG. 2 schematically shows a process of separating total proteins including a microstructure including a olfactory tissue and 7-membrane penetration proteins in a fruit fly using a Drosophila micro-organ separator.

3. Immunoblotting ( immunoblotting )

In order to visualize the 7-membrane penetration protein through immunoblotting in the pulverized Drosophila microstructure, the pulverized microstructure is immediately mixed with SDS buffer (GFP-Trap®_A protocol; ChromoTek, Germany) Samples were prepared by mixing the SDS buffer with the 2X ratio to the tissue lysate obtained in the above 2. using the dissolution buffer. The lysates mixed with the SDS buffer were heated at 50 ° C for 30 minutes using an Eppendorf ThermoMixer ™ C (Hamburg, Germany) while shaking at 500 rpm.

For the sample used in Figure 4, the tissue lysates were dispensed in equal volumes into 6 tubes, and each tube was heated at 50, 55, 60, 65, and 70 ° C for 30 minutes or at 95 ° C for 10 minutes And heated.

All the wells were loaded with 30 μg of total protein and the total protein electrophoresed on 10% SDS-PAGE was transferred to a PVDF membrane (Merck, IPVH00010) and the PVDF membrane was incubated with primary antibody Lt; / RTI > overnight. GFP was detected with anti-GFP (ThermoFisher, A-11122) antibody diluted to 1 / 5,000 in 5% skim milk / TBST and antiglyceraldehyde 3-phosphate dehydrogenase (abcam, ab125247 ) Was used as a loading control. The second antibody was contacted with the anti-rabbit HRP-conjugated antibody (Merck, AP132P) or 1 / 5,000 diluted anti-rabbit antibody (ThermoFisher, 31430). In all experimental procedures, the loading control was first stripped (Atto, WSE-7240) and then contacted with the antibody.

4. Immunoprecipitation ( Immunoprecipitation , IP) and on- Bead Diez cloth (on-bead digestion)

All immunoprecipitation-mass spectrometry samples were prepared in a 1% (w / v) DDM dissolution buffer, prepared the same day as mass spectrometry (MS) grade water. The tubes were filled with 50 μl of GFP-Trap® A bead slurry, equilibrated three times with 500 μl of lysis buffer, and 5 mg of protein was added. The binding reaction between the protein and the bead was carried out for 2 hours on a rotating platform at room temperature. After 2 hours, the beads were collected, washed five times with 1 ml of lysis buffer, and washed once with 1 ml of Tris-HCl (pH 8.5). The on-bead digest cloth utilized a method known in the art (Mol Cell Proteomics. 2011 May; 10 (5): M110 006460).

5. Mass spectrometric analysis (MS analysis)

After on-bead digestion, salts were removed from the digested peptides using STAGE-Tips filled with C18 disks (3M, 2215) and dried in a vacuum concentrator. For mass analysis, 0.1% formic acid was added to the dried peptide to acidify it. Peptide assays were performed with an EASY-nLC 1000 solution (Thermo Fisher) coupled to a Q-Exactive mass spectrometer (Thermo Fisher) equipped with a nanoelectrospray ion source. The peptides were separated by a 15-cm reversed phase column with an internal diameter of 75 [mu] m filled with 100 [mu] M C18 beads of 3 [mu] m size. Peptides were analyzed for 130 minutes at a flow rate of 350 nL / min by applying a concentration gradient from 9.5% to 36.5% of a solvent in which 0.1% formic acid and acetonitrile were mixed. To automatically switch between full-scan MS and tandem mass spectra (tandem MS), the mass spectrometer was operated in a data-dependent mode. The full-scan mass spectra were collected at Orbitrap (300-1800 m / z) using an automated gain control (AGC) value of 1,000,000 at 70,000 resolution. Twelve of the strongest ions in the full-scan were separated using AGC 500,000 at a resolution of 17,500. The isolated ions were fragmented in a high collision dissociation cell by collisionally-induced dissociation with a normalized collision energy of 27% and a 2 m / z isolation width.

6. Data Analysis

The raw date file is the UniProt Drosophila We searched using MaxQuant (version 1.6.0.1) in the melanogaster database (UP000000803; Mar. 15, 2017). The parameters used for the search were tryptic digestion, up to two unmethylated cysteines, fixed carbamidomethyl modifications of cysteine, variable oxidation of methionine, A variable acetylation of protein N-termini of the protein N-terminus was applied. The mass tolerance of the precursor ions was 4.5 ppm and the mass tolerance of the fragmented ions was set at 20 ppm. To confirm the false discovery rate (FDR), a target database was inverted to create a decoy database. The detection rate of protein and peptide levels was fixed at 1%, and only proteins identified as one or more unique peptides were selected.

Experiment result

1. Tissue collection and protein separation

In order to perform immunoblotting efficiently, Orco protein (EGFP :: Orco) functioning normally in vivo and fused with EGFP at the N-terminus was used. In addition, EGFP :: Orco was expressed in the Orco-null mutant ( Orco ¹ ) to prevent the competition between the wild-type Orco protein and EGFP :: Orco for the interacting protein.

In order to isolate the Orco interactome from the olfactory tissue of the Drosophila, preference was given to the olfactory collection method. First, a microporous microfissure separation device was devised to directly collect olfactory tissue in a 50 ml tube. The inventor invented a Drosophila microstructure separator can be used to collect the olfactory tissue much more quickly and easily than by manually dissecting the tissue (the antenna or head of the Drosophila) by hand. In addition, as shown in FIG. 3, it can be seen that the microstructure collected using the Drosophila microstructure separation apparatus contains relatively more EGFP-labeled Orco than the hair tissue.

In the olfactory tissue of the microstructure separated by sieving, the axon of the OSN was destroyed, so the separated protein was solubilized as soon as possible.

Various tissue grinding methods were considered to ensure that the 7-membrane penetrating protein (especially Orco) present in the fine and hard microcapsules of the collected microstructure is more efficiently released into the lysis buffer. As a result, it was confirmed that when the iron bead tissues were homogenized as shown in Table 1, the proteins were consistently separated in a similar amount.

tool Repeat order Protein concentration (/ /)) Standard Deviation
Plastic rods
(Plastic pestle) One 0.89
0.34 2 1.12 3 1.69 4 1.29
Iron bead
(Steel beads) One 1.47
0.16 2 1.34 3 1.69 4 1.65

Next, after cleavage of the olfactory tissues, a buffer containing various surfactants to optimize conditions suitable for immunoblotting and mass spectrometry of the 7-membrane penetrating protein Orco, and a buffer containing various surfactants such as EGFP :: Orco The possibility of solubilization of As a result, all the surfactants except Tween 20, CHAPS and CHAPSO were found to solubilize EGFP :: Orco as shown in FIG. 4A. Based on these results, lysis buffers containing 1% DDM were used for all future experiments including mass spectrometry. In addition, as shown in FIG. 4B, when the protein sample was heated in the presence of SDS and? -Mercaptoethanol, the solubilization degree of EGFP :: Orco solubilized by DDM was decreased as the temperature was increased. Therefore, it was confirmed that the solubilization of EGFP :: Orco at 50 was optimized for immunoblotting.

2. IP-MS ( Immunoprecipitation -mass spectrometry)

In order to classify non-specific proteins bound to beads and EGFP markers, parallel IP experiments were performed with lipid-conjugated GFP (myristoylated GFP, myr :: GFP). Anti-GFP Nanobodies; that (the variable domain of the Camelidae heavy chain antibody Harmsen and De Haard, 2007) was performed using an IP connected to agarose beads.

The proteins immunoprecipitated with Orco were then identified by liquid chromatography-tandem MS (LC-MS / MS) analysis. In order to identify immunoprecipitated proteins with Orco, on-bead digestion was performed by analyzing tryptic peptide (tryptic peptide) in immune precipitation sample bound to the nano-body with MS Method.

3. Data processing and analysis

The raw mass spectrometric data were obtained from Uniprot Drosophila We searched for the melanogaster database using MaxQuant. The first 1,057 identified proteins were obtained and this list was ranked according to the experimental-to-control iBAQ value ratios of the experimental and control groups. In order to select proteins that are likely to interact with Orco, only iBAQ values were detected in all biological replicates of the experimental group, while the control iBAQ value was zero. As a result, four proteins were expected to interact with each other with a high probability, as shown in Table 2 below. According to the results in Table 2, it can be seen that the immunoprecipitated Orco was separated in the largest amount. This shows that the 7-membrane penetration protein can be effectively separated when the interaction product is obtained using the process of FIG. 2 and the specific IP-MS method described above.

Gene name The protein of interest Experiment iBAQ value Representative Cellular Component Ontology Orco Odorant receptor coreceptor 5.40 x 10 ⁸ Dendrites, platelets CG34309 (Undefined) 5.47 x 10 ⁷ Membrane protein Or92a Odorant receptor 92a 1.95 x 10 ⁷ Dendrites, platelets CG5382 Zinc finger protein-like 1 homolog 2.43 x 10 ⁶ Intracellular membrane system

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (5)

Separating the olfactory olfactory tissue;
Crushing the separated olfactory tissue; And
A method for separating 7-membrane penetrating protein from a olfactory olfactory tissue comprising the step of adding buffer to the crushed olfactory tissue and heating.
The method of claim 1, wherein the step of grinding the olfactory tissue uses iron beads.
The method of claim 1, wherein the buffer comprises a surfactant.
The method of claim 3, wherein the surfactant is selected from the group consisting of Triton X-100, Nonidet P-40, Octyl-bD-glucopyranoside, n-Dodecyl b-Dmaltoside and Zwittergent 3-16.
2. The method of claim 1, wherein the heating is performed at a temperature of from 40 to 60 < 0 > C.

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