JP2009183256A - Method of measuring protein expression using cultured cell on real-time basis - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method of easily and accurately measuring the expression level of a target protein on a real-time basis under a normal cell culturing condition using a cultured cell without recovering nor destroying the cell. <P>SOLUTION: The method of quantitatively measuring the expression level of the target protein in the cultured cell comprises: (1) a process of culturing a cell capable of expressing a fusion protein formed of a target protein and a fluorescent protein in a culture tank; (2) a process of measuring the fluorescence intensity of the fusion protein expressed in the cell cultured in the process (1), without crushing the cultured cell nor transferring the same to the outside of the culture tank; and (3) a process of calculating the expression level of the target protein using the fluorescence intensity as an indicator. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for measuring protein expression. More specifically, the present invention relates to a method for quantifying the amount of a target protein expressed in cultured cells using the measured fluorescence intensity as an index.

  In order to conduct biochemistry, physical chemistry, structural biology research, etc. of proteins, it is necessary to establish a condition for mass production (expression) using various cells because a large amount of the protein to be studied is required. It is. In particular, many eukaryotic proteins, including humans, are produced using animal cultured cells such as insect cultured cells and mammalian cultured cells. Especially, membrane proteins are expressed in animal cultured cells. It is essential. However, when examining the amount of the target protein expressed in these cells, it is necessary to collect the cultured cells, crush them, and quantify them using various experimental techniques. A large amount of sample and labor were required for the optimization.

In recent years, the target protein to be studied is expressed as a green fluorescent protein (GFP) fusion protein, and the fluorescence measurement of cells (E. coli and yeast in previous reports), and then the cells are disrupted and subjected to electrophoresis and gel filtration chromatography. Thus, a technique for estimating the expression level of the target protein by performing fluorescence measurement has been reported (see Non-patent documents 1 to 3). However, in the conventional method, it is necessary to measure the fluorescence after collecting the cells after culturing, and it is impossible to measure the protein expression level in the cell culture in the process of producing the protein in real time. Therefore, in order to measure changes over time in protein expression, it is necessary to prepare multiple samples, incubate them for different times, and then collect them, and there is a risk of errors due to variations in cell growth between samples. there were. Furthermore, microorganisms such as Escherichia coli and yeast that have been reported to directly measure fluorescence intensity from cells have been measured because the cells are opaque and excitation light and fluorescence for fluorescence intensity measurement are absorbed by the cells. Inaccurate, and in order to perform an accurate measurement, a great deal of labor was required such as crushing the cells and observing the fluorescence by electrophoresis or gel filtration chromatography.
Drew et al., 2006, Nat.Methods 3, 303-313. Kawate et al., 2006, Structure 14, 673-681. Newstead et al., 2007, Proc. Natl Acad. Sci. USA 104, 13936-13941.

  An object of the present invention is to provide a method for easily and accurately measuring the expression level of a target protein by cultured cells in real time without recovering and destroying the cells under normal cell culture conditions.

  In order to solve the above problems, the present inventors have paid attention to a method of measuring the expression level of the protein by expressing the target protein as a GFP fusion protein and measuring the fluorescence using a plate reader. Repeated examination. As a result, an expression vector for expressing the target protein whose expression level is to be examined as a GFP fusion protein was constructed, and a gene introduced into animal cultured cells was used in a multiwell plate in the same manner as in normal cell culture. We found that it is possible to estimate the expression level of the target protein during the culture by measuring the fluorescence of the cultured multi-well plate as it is using a plate reader. The invention has been completed. That is, the present invention is as follows.

[1] A method for quantifying the amount of a target protein expressed in cultured cells, comprising the following steps:
(1) culturing a cell capable of expressing a fusion protein of a target protein and a fluorescent protein in a culture vessel;
(2) measuring the fluorescence intensity of the fusion protein expressed in the cells cultured in (1) without disrupting the cells and without transferring them outside the culture vessel; and (3) the measured fluorescence intensity. Calculate the expression level of the target protein as an index.
[2] The method according to [1] above, wherein the fluorescence intensity measurement is a downward measurement.
[3] The method according to [1] above, wherein the fluorescence intensity is measured without removing the lid of the culture vessel.
[4] The method of the above-mentioned [1], wherein the bottom area per well of the culture container exceeds 0.32 cm ² .
[5] The method according to [1] above, wherein the fluorescence intensity is measured by multipoint measurement.
[6] The method of the above-mentioned [5], wherein a region exceeding 70% of the bottom area of the region not including the container edge of the culture vessel is subjected to multipoint measurement.
[7] The method according to [1] above, wherein the fluorescence intensity in a culture vessel containing only the culture medium and not containing cells is also measured for normalizing the fluorescence intensity.
[8] The method of the above-mentioned [1], wherein the cell is a cultured animal cell.
[9] The method according to [1] above, wherein the fluorescence intensity is continuously measured in the same cell culture tank.

  In the method of the present invention, since it is not necessary to collect cells or open / close the lid of the culture plate in order to measure the expression of the target protein, the cultured cells are kept as they are without exposing them to the risk of contamination. The expression of the target protein can be evaluated in real time, and the protein expression over time can be measured from the same sample in one culture. This makes it possible to easily determine the expression time, which is an important factor for establishing protein expression conditions. In addition, compared to the direct fluorescence measurement from microbial cells reported so far, the use of transparent animal culture cells suppresses the absorption of excitation light and fluorescence, and the fluorescence intensity increases the protein expression level. It is thought that it is accurately reflected. For this reason, it is possible to estimate the expression level by direct measurement of the cells without performing the quantification, which is performed using electrophoresis, gel filtration chromatography, or the like after recovering and crushing the cells. Because of these simplicity, this method is also suitable for high-throughput analysis in which the expression levels of many types of target proteins are compared at once.

The present invention provides a method for quantifying the amount of a target protein expressed in cultured cells. The method includes the following steps:
(1) culturing a cell capable of expressing a fusion protein of a target protein and a fluorescent protein in a culture vessel;
(2) measuring the fluorescence intensity of the fusion protein expressed in the cells cultured in (1) without disrupting the cells and without transferring them outside the culture vessel; and (3) the measured fluorescence intensity. Calculate the expression level of the target protein as an index.

  The target protein quantified by the method of the present invention is an arbitrary protein whose expression level in cultured cells is to be examined. The origin may be any origin such as prokaryotic origin or eukaryotic origin, and the type of protein such as secretory protein, membrane protein, cytoplasm or protein localized in the nucleus is not limited.

  As long as the fluorescent protein is suitable for fusion protein expression, any one used in the technical field can be suitably used. For example, it is derived from Owan jellyfish such as GFP, YFP, CFP, BFP, and Venus. Examples include fluorescent proteins and analogs thereof, Renilla-derived fluorescent proteins and analogs thereof, coral-derived fluorescent proteins such as DsRed, HcRed, AsRed, ZsGreen, ZsYellow, AmCyan, AcGFP, and Kaede, and analogs thereof. The determination of the fluorescent protein to be used can be made in consideration of various factors such as the expression vector to be used and cultured cells, or the price, and this is within the ordinary technical knowledge of those skilled in the art. Usually, GFP derived from Aequorea or its analog (eg, EGFP) is preferably used. As will be described later, in the method of the present invention, in order to incorporate a nucleic acid encoding a fusion protein of a target protein and a fluorescent protein into an appropriate expression vector, a nucleic acid encoding the fluorescent protein is required. A vector commercially available from Clontech may be used.

  In order to produce a cell capable of expressing a fusion protein of a target protein and a fluorescent protein, an appropriate expression vector for the fusion protein may be constructed and introduced into an appropriate cell. As said cell, all the cells normally used for protein manufacture can be used without limitation. In the method of the present invention, since the fluorescence intensity from the cells is measured without disrupting the cells, it is more transparent than microbial cells such as E. coli and yeast from the viewpoint of enabling more accurate measurement of the fluorescence intensity. It is preferable to use animal culture cells having the property of adhesion culture. Animal cell cultures are derived from mammals (eg, hamsters, mice, monkeys, humans, etc.), birds (eg, chickens, turkeys, etc.), reptiles (eg, snakes, etc.), amphibians (eg, frogs), fish, Examples include insects (eg, mosquitoes, flies, moths, etc.), but mammalian cells or insect cells are usually preferred. Examples of cultured cells derived from mammals include CHO cells, BHK cells, NS0 cells, SP2 / 0 cells, COS cells, HEK293 cells, and the like. On the other hand, examples of insect-derived cultured cells include Sf9 cells, Sf21 cells, and High Five cells.

The expression vector for the fusion protein should contain a base sequence encoding the fusion protein of the target protein and the fluorescent protein and be capable of expressing the fusion protein in the introduced cell.
A fusion protein expression vector can be produced, for example, by excising a DNA fragment encoding a target protein and a DNA fragment encoding a fluorescent protein and ligating them downstream of a promoter in an appropriate expression vector. . At that time, the target protein and the fluorescent protein need to be synthesized as one fused protein. The method may be performed by referring to the manufacturer's instructions when using a base sequence encoding a commercially available fluorescent protein, or according to a conventional method described in many standard laboratory manuals.
Expression vectors to which the fusion protein genes are linked include plasmids derived from E. coli (eg, pBR322, pBR325, pUC12, pUC13), plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, pC194), yeast-derived plasmids (eg, In addition to bacteriophages such as pSH19, pSH15) and λ phage, animal viruses such as retrovirus, vaccinia virus, baculovirus, and the like, pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo and the like are used.
The promoter may be any promoter as long as it is appropriate for the cultured cells used for gene expression. For example, when using animal cells, SRα promoter, SV40 promoter, LTR promoter, CMV promoter, HSV-TK promoter and the like can be mentioned. Of these, the CMV (cytomegalovirus) promoter, SRα promoter and the like are preferably used. When insect cells are used, IE1 promoter, polyhedrin promoter, P10 promoter and the like can be mentioned. Of these, the IE1 promoter is preferably used.
In addition to the above, an expression vector containing an enhancer, a splicing signal, a poly A addition signal, a selection marker, an SV40 replication origin, or the like can be used as desired. Examples of the selection marker include dihydrofolate reductase gene [methotrexate (MTX) resistance], ampicillin resistance gene, neomycin resistance gene [G418 resistance] and the like. Moreover, you may add the signal arrangement | sequence suitable for the cultured cell to be used to the N terminal side of the target protein as needed.
A transformant can be produced using an expression vector containing a DNA encoding the fusion protein thus constructed.

  The introduction of the expression vector into the cultured cell may be for the purpose of stable integration into the cell chromosome or may be a transient introduction. The introduction may be performed by a method known per se, and examples thereof include transfection such as electroporation, lipofection, calcium phosphate method (CaPi), polyfection method (PEI), and calcium chloride method (culfection). Such methods are described in many standard laboratory manuals.

Methods for culturing cells are known in the art (see, for example, Tissue Engineering Methods and Protocols, Morgan and Yarmush (eds.), Humana Press, Inc., Totowa, NJ, 1999). As one skilled in the art will recognize, the conditions under which cells are cultured will vary with the type of cell. The conditions include the temperature of the environment, the culture vessel containing the cells, the composition of various gases (eg, CO ₂ ) constituting the cell culture atmosphere or environment, the medium in which the cells are maintained, the components and pH of the medium, the cells The density at which the medium is maintained, a schedule that requires the medium to be replaced with a new medium, and the like. These parameters are often known in the art or can be determined empirically.
When the cultured cells are animal cells, examples of the medium include MEM medium [Science, 122, 501 (1952)] containing about 5 to 20% fetal bovine serum, DMEM medium [Virology, 8, 396 ( 1959)], RPMI 1640 medium [The Journal of the American Medical Association, 199, 519 (1967)], 199 medium [Proceeding of the Society for the Biological Medicine, 73, 1 (1950)] and the like. The pH is preferably about 6-8. Culture is usually performed at about 30 to 40 ° C. for about 15 to 60 hours, and aeration and agitation are added as necessary. In addition, when the cultured cells are insect cells, a medium obtained by appropriately adding an additive such as 10% bovine serum immobilized in Grace's Insect Medium (Nature, 195, 788 (1962)) is used. The pH of the medium is preferably adjusted to about 6.2 to 6.4. The culture is usually performed at about 27 ° C. for about 3 to 5 days, and aeration and agitation are added as necessary.

The culture vessel for carrying out the cell culture holds a medium for culturing cells, and measures fluorescence intensity without transferring the cultured cells to the outside of the culture vessel, as will be described later. There is no particular limitation as long as it is possible. As long as the above conditions are satisfied, for example, a microplate, a culture slide provided with a chamber or a plurality of chambers, a cover glass, a cup, a flask, a tube, a roller bottle, a spinner bottle, etc. can be used as a culture container. Since the fluorescence intensity measurement in the method of the present invention is usually performed using a microplate reader, a microplate is preferably used.
In the case of the microplate, if the bottom area of each well is too small, reproducible cell growth cannot be maintained, the culture medium may be dried up, and fluorescence intensity measurement. There are disadvantages such as not allowing multi-point measurement, and therefore there is a tendency that reproducible results cannot be obtained, and therefore it is necessary to have a sufficiently large bottom area. Specifically, for example, a commercially available 96-well plate having a bottom area exceeding 0.32 cm ² which is the bottom area is preferably used. The upper limit of the bottom area is not particularly limited as long as measurement of fluorescence intensity is allowed, but it is usually 500 cm ² or less. Here, the bottom area is more preferably 0.5～20.0Cm ^2, more preferably 1.8~9.6cm ^2. Similarly, culture vessels other than microplates need to have a sufficient bottom area.
The shape of the well of the microplate includes a flat bottom, a U bottom, and a V bottom, but for the method of the present invention, the measurement focal height in the microplate reader is usually fixed. Therefore, a flat bottom in which cells are distributed at the same height when adhesion culture is performed is preferable.
Examples of the material for the bottom of the well of the microplate to which cells adhere during adhesion culture include polystyrene, white polystyrene, polyethylene, natural polypropylene, glass-filled polypropylene, plastic such as Valex, or glass such as quartz glass or borosilicate glass. In addition, a plastic microplate, particularly a polystyrene microplate, can be suitably used in the method of the present invention because it has excellent cell adhesion during culture and is inexpensive. Furthermore, from the request for fluorescence observation, the bottom surface must be transparent, and those made of polystyrene satisfy this requirement.
Commercially available microplates such as Corning's multiple well plates are usually used, and in order to satisfy the conditions regarding the bottom area, wells having 24 holes or less (eg, 6 holes, 12 holes, 24 holes, etc.) are used. A holding microplate can be preferably used.
The microplate body includes transparent, white, and black ones. White or black microplates are generally more expensive than transparent microplates, and these commercially available microplates are also commercially available. Considering that the material of the bottom surface of the plate is not necessarily suitable for cell adhesion culture, a transparent material is preferable.
From the above, in the method of the present invention, an example of a microplate that is particularly preferably used is a 6-hole or 24-hole polystyrene flat-bottom transparent microplate.

  In the method of the present invention, the fluorescence intensity of the fusion protein needs to be measured without destroying the cultured cells and without transferring them to the outside of the culture vessel. Any fluorescence intensity measurement method used in the art can be used as long as such a condition is satisfied, but fluorescence intensity measurement with a microplate reader is preferable from the viewpoint of sensitivity, accuracy, and simplicity. Examples of the microplate reader include a Varioscan flash manufactured by Thermo Fisher Scientific. Moreover, those skilled in the art can measure the fluorescence intensity with reference to a manual attached to the measuring instrument to be used.

  The measurement of fluorescence intensity with a microplate reader can be performed by multipoint measurement, which obtains the average value of fluorescence intensity measured at a plurality of points in a culture vessel as the fluorescence intensity in the entire vessel. It is preferable from the viewpoint of minimizing. In that case, it is preferable that a region exceeding 70% of the bottom area of the region not including the container edge of the culture vessel (hereinafter also referred to as “measurement region”) is subjected to multipoint measurement. There is no particular upper limit for the bottom area of the region to be subjected to multipoint measurement, but considering the time required for measurement, 95% of the measurement region is preferable, 90% is more preferable, 85% is further preferable, and 80% is particularly preferable. . Here, the container edge is an inside of a container whose distance from the container edge is within a range of a distance having a larger value of 3 mm or twice the beam width of the measurement light beam of the measuring device. Means the part. In addition, as described above, the measurement of the fluorescence intensity in the present invention is performed without transferring the cultured cells to the outside of the culture container. Therefore, from the viewpoint of preventing contamination of the culture container, the lid of the culture container is not removed. It is preferable to measure the fluorescence intensity. When measuring fluorescence intensity from a culture vessel with a lid, the upper measurement, that is, the fluorescence intensity measurement from a certain direction of the lid, can be used in control without a fluorescent substance because of irregular reflection due to excitation light and fluorescence lid. Since there is a problem that a high fluorescence intensity value is exhibited and thus the signal-to-noise ratio is low, in the measurement of the fluorescence intensity in the present invention, it is possible to measure the fluorescence intensity from the bottom side of the culture vessel, that is, from the bottom side of the culture vessel. preferable.

  As described above, since the measurement of the fluorescence intensity in the present invention is performed without crushing the cells and without transferring them to the outside of the culture vessel, the fluorescence intensity can be continuously measured in the same cell culture tank. Is possible. Therefore, protein expression over time can be measured from the same sample in a single culture, and the expression time, which is an important factor for establishing protein expression conditions, can be easily determined.

As described above, the method of the present invention includes calculating the expression level of the target protein using the measured fluorescence intensity as an index. In order to calculate the expression level of the target protein, the measured fluorescence intensity is preferably normalized by the fluorescence intensity derived from an appropriate control. As such a control, it is preferable to use a culture vessel that does not contain cultured cells and contains only the same type of culture medium used for cell culture, from the viewpoint of obtaining highly reproducible results. Especially when using multi-well microplates, use fluorescence intensity measurements from wells filled with the same type and volume of culture medium as used for sample culture on the same culture plate of the fluorescence intensity measurement sample. Is preferred. In that case, the fluorescence intensity F in each well of the microplate is:
Relative fluorescence intensity = (F−F ₀ ) / F ₀
(Where F ₀ is a measurement of fluorescence intensity from a well filled with culture medium in the same culture plate as the measurement sample);
It is normalized by the equation Using the relative fluorescence intensity thus obtained as an index, the expression level of the target protein can be quantified.

  EXAMPLES Hereinafter, although an Example etc. demonstrate this invention further in detail, this invention is not restrict | limited to the following Example etc. at all.

Example 1
(1) Construction of Expression Vector As described below, a vector for testing two types of protein expression, cytoplasmic expression and secretory expression, in insect cultured cells was constructed (FIG. 1). The test vector pCGFP_SF for cytoplasmic expression is a gene for a modified GFPuv (a mutant of GFP having an excitation maximum in the UV region) having a multicloning site, a thrombin cleavage site and a hexa-histidine tag, and a vector pCGFP-BC (Eric Gouaux Providing courtesy of the professor Non-patent document 2) Amplified by a conventional PCR method using a template and treating the fragment with the restriction enzyme DraIII, the NcoI site of the pIEx4 vector (Novagen) (blunted) And between DraIII sites. The vector pCGFP_SF + AKH, which is a test vector for secretory expression, designed an oligonucleotide primer so that an adipokinetic hormone (AKH) signal sequence, which is an insect-derived secretory signal sequence, can be linked upstream of the multicloning site of the pcGFP_SF vector ( The AKH signal sequence was designed with reference to the sequence contained in the pIEx-5 vector (novagen)), and was constructed in the same manner as the pcGFP_SF vector using a normal PCR method (FIG. 1). In order to construct an expression vector for the test protein in Example 1 below, human-derived taste receptor ligand binding region T1r1 (456 residues, protein A), T1r2 (454 residues, protein B) from which the secretion signal was removed , T1r3 (458 residues, protein C) and a rat metabotropic glutamate receptor 1 ligand binding region gene (522 residues, protein D) containing a secretion signal were subcloned into the pCGFP_SF vector to obtain a C-terminal GFP fusion .

(2) Expression system For evaluation of real-time detection of recombinant protein expression in the present invention, Sf9 cells were used in a transient protein expression system. The Sf9 expression system is one of the most commonly used systems for recombinant protein expression in the field of structural biology research. In contrast to expression systems in microorganisms such as E. coli and yeast cells, Sf9 cells, like other animal cultured cells, are transparent and allow more accurate measurement of the fluorescence intensity of expressed GFP in living cells. Cells were subjected to monolayer culture (cells adhere to the bottom of the culture plate), the most standard and widely applied culture method for normal animal cells.
Specifically, the expression of the recombinant protein was performed as follows. The expression vector was transfected into Sf9 cells (GIBCO) using Insect Genejuice (Novagen) according to the manufacturer's protocol. Transfected cells were incubated in Sf900III SFM (GIBCO) for 48-120 hours at 27 ° C. in various types of tissue culture plates to allow transient recombinant protein expression. Typically, Sf9 cells from confluent monolayer cultures have a density of 1.0-1.6 × 10 ⁶ cells / well in each well in a 6-well tissue culture plate (6 Well Clear TC-Treated microplates, Costar). The wells were then filled with 1 ml Sf900III SFM containing 10 μl Insect Genejuice and 2 μg plasmid. One of the wells in the culture plate was filled with an equal volume of Sf900III SFM and used as a control (background).

(3) Types of culture plates To test various cell culture plates, cells were incubated in three different types of culture plates, 6, 24 and 96 well plates after transfection. Of these plates, 96-well plates gave little reproducible results. The wells of the 96-well plate were too small to maintain reproducible cell growth and often faced dry culture media. Furthermore, the single well of the 96-well plate allows only one point measurement due to its small size, which further increases the measurement error during detection (data not shown). On the other hand, cells cultured in 6-well and 24-well plates show stable cell growth, and the size of these wells allows multipoint measurements and averages values in various regions of a single well Therefore, the state of the number of cells in the well can be reflected more accurately. Next, a conventional transparent culture plate of polystyrene (6 Well Clear TC-Treated Microplates, Costar) is used as a glass bottom black culture plate (EZview glass bottom culture plate LB, 6 Well, Iwaki). However, the detected S / N ratio (relative fluorescence intensity values) between these two plates was approximately the same at two different time points for both cytoplasmic and secreted protein expression (FIG. 5). In FIG. 5, SF_top and SF_bottom represent the results of the upward or downward measurement of the cells transfected with pCGFP_SF, respectively, and AKH_top and AKH_bottom are the upward or downward measurement of the cells transfected with pCGFP_SF + AKH. The results are shown respectively. Considering that black culture plates are expensive and not necessarily suitable for cell culture, the use of conventional 6-well or 24-well polystyrene transparent tissue culture plates is preferred and sufficiently accurate for real-time fluorescence detection. It is thought to give sex.

(4) Determination of measurement parameters for fluorescence detection using a microplate reader For accurate measurement of the fluorescence intensity of GFPuv in living Sf9 cells , for detection with a microplate reader Varioskan Flash (Thermo Fisher Scientific) Measurement parameters were determined. Initially, an attempt was made to find a suitable method for normalization of the measured fluorescence intensity. Two conditions were compared: untransfected Sf9 cells (treated with lipofection reagent without using a plasmid) and culture medium to assess which served as an appropriate control for the measurement. The fluorescence intensity values under these conditions were in a similar range, but using the culture medium as a control resulted in higher reproducibility than using untransfected Sf9 cells. (Data not shown). Untransfected Sf9 cells sometimes exhibit much higher cell growth and fluorescence intensity than transfected cells, resulting in sample relative fluorescence intensity values that are unsuitably small for use in normalization. . Therefore, culture medium was employed as a control.
In Example 1 and Comparative Example 1 below, the fluorescence intensity derived from GFPuv expressed in cell growth in a tissue culture plate was measured using a microplate reader Varioskan Flash (Thermo Fisher Scientific) using an excitation wavelength of 395 nm and a fluorescence wavelength of 507 nm. ). Typical measurement conditions were set for multipoint measurement (circular area from the center of the well and 80% of the measurement area) with a measurement time of 500 ms and a bandwidth of 12 nm. In this experiment, a test for multipoint measurement in an area exceeding 80% of the measurement area of the well was not performed. The fluorescence intensity F measured in each well of the culture plate is:
Relative fluorescence intensity = (F−F ₀ ) / F ₀
(Here, F ₀ represents the fluorescence intensity of the culture medium filled in the well in the same culture plate of the measurement sample)
It normalized with the equation.

  Next, various measurement parameters for fluorescence detection with a microplate reader were tested. From the viewpoint of signal-to-noise ratio (S / N ratio) (relative fluorescence intensity value), the following conditions gave the best results; the measurement method was set at multiple points, the measurement time was 500 ms, and the bandwidth was 12 nm. Met. Of the areas tested in multipoint measurements, a circular area from the center of the well covering about 70-80% of the bottom area of the well measurement area gave the best results (data not shown). In addition, two fluorescence detection methods, upper measurement and lower measurement, were compared. The results showed that the lower and upper measurements of the plate with the top cover of the culture plate removed gave similar values (FIG. 3). However, in the case of reading a lidded plate, the upward measurement showed a high background (high fluorescence intensity value in the control), resulting in a low signal-to-noise ratio (FIG. 4), which is probably excited. This is because of irregular reflection by light and fluorescent lid. In contrast, the lower measurement showed similar values compared to the measurement of the plate without lid (FIGS. 3 and 4). In FIGS. 3 and 4, SF_top and SF_bottom represent the results of the upper measurement or the lower measurement of the cells transfected with pCGFP_SF, respectively, and AKH_top and AKH_bottom represent the upper measurement of the cells transfected with pCGFP_SF + AKH or The results of the downward measurement are shown respectively. Removing the lid of the plate during cell culture greatly increases the risk of contamination. Therefore, the best method for measurement is to use the down measurement and measure the culture plate without removing the top cover. This method makes it possible to keep the culture intact and to use a single test culture to follow the time course of protein production during cell culture. These measurement parameters work well in detecting the amount of recombinant protein by both cytoplasmic and secretory expression (FIGS. 3 and 4). For cytoplasmic expression of GFP using the vector pCGFP_SF, the observed time course of protein production was in good agreement with that normally seen using the Sf9 expression system. For the secreted expression of GFP using the vector pCGFP_SF + AKH, the observed increase in fluorescence intensity with time of incubation corresponds to the accumulation of the produced protein in the culture medium, although the detected value is smaller than that of cytoplasmic expression It seems to be doing.

(5) Results The above protocol was applied to four different test proteins A, B, C, and D. Of these test proteins, the results for A, B, and C are shown in FIG. This result indicated that this system functions well not only for GFP alone but also for these proteins expressed by GFP fusion in both cytoplasmic expression and secretory expression. The observed fluorescence intensity of the GFP fusion test protein was different from that of GFP alone, and a difference was also observed between test proteins A, B, and C under the same expression conditions. From this, the fluorescence intensity from each sample (and hence the amount of recombinant GFP fusion protein expression), as suggested in previous studies (see Non-Patent Documents 1 to 3), determines which test protein is in Sf9 cells. Therefore, it was suggested that it is considered not to depend on the expression of GFP alone as a reporter protein. This result suggests that the method of the present invention is applicable to normal proteins and is useful for high-throughput sample screening.

Comparative Example 1: Verification of Quantitative Property of the Method of the Present Invention For verification of the quantitative property of the real-time fluorescence detection method in the present invention, the results were reported using a fluorescence detection gel filtration chromatography (FSEC), which is a reported quantitative method. (See Non-Patent Document 2). In the FSEC method, the amount of GFP fusion protein can be assessed using the elution peak area from gel filtration chromatography detected by fluorescence. Cell debris supernatants (in the case of cytoplasmic protein detection) and culture media (in the case of secreted protein detection) from the same sample used for real-time detection with a plate reader were analyzed by FSEC.
The specific method of the comparison is as follows. After protein expression at the appropriate time and fluorescence detection with a microplate reader, Sf9 cells and culture medium were harvested separately. The cells were washed twice with suspension buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, and 5 mM KCl) and suspended in 300 μl of the same buffer. The cells were then sonicated on ice using Handy Sonic (TOMY SEIKO) at output 5 for 2-3 minutes. The sample was centrifuged at 15 k rpm for 30 minutes at 4 ° C. and the supernatant was filtered through Ultrafree-MC (Millipore). The culture medium was concentrated with Vivaspin (10k MWCO) (VIVASCIENCE), diluted to 300 μl with suspension buffer, and filtered with Ultrafree-MC.
Using a running buffer (20 mM Tris-HCl pH 7.5, 200 mM NaCl) on a Superose 6 10/300 GL column (GE Healthcare) connected to an AKTA explorer instrument (GE Healthcare), a flow rate of 0.5 to 1.0 ml / FSEC analysis was performed by loading 200 μl of sample per minute. The elution of gel filtration chromatography was detected using a fluorometer RF-10AXL (Shimadzu) using an excitation wavelength of 395 nm and a fluorescence wavelength of 597 nm. The amount of expressed GFPuv protein was estimated by integrating the range of elution peaks derived from GFPuv.

  The result of the comparison is shown in FIG. The FSEC results were almost consistent with those determined by real-time fluorescence detection in the present invention. Significantly, the detection range of the microplate reader included both cells attached to the bottom of the culture plate and the culture medium on top of the cells, but was transfected with pCGFP_SF and pCGFP_SF + AKH. The cell results reflected well the amount of cytoplasmic protein and secreted protein, respectively, which are the samples of interest.

[Acknowledgments]
Special thanks to Prof. Eric Gouaux (currently the University of Oregon Health Sciences Volum Institute) and Columbia University for providing the pCGFP-BC vector used to create the expression vector for testing in the Examples . Part of this research is supported by the Ministry of Education, Culture, Sports, Science and Technology and the target protein research program “Development of innovative support methods for membrane protein crystallization”.

It is a figure which shows the design of the expression plasmid for Sf9 cells used in the Example. It is a figure which shows the fluorescence of GFPuv transiently expressed in the Sf9 cell observed with the fluorescence microscope. FIG. 6 shows GFPuv fluorescence from transfected Sf9 cells in a 6-well culture plate without lid, detected by a microplate reader. The vertical axis represents the relative fluorescence intensity value. From the left of each column, it corresponds to the relative fluorescence intensity at the time of 48 hours, 72 hours, 96 hours, 120 hours, and 144 hours from the start of culture. FIG. 5 shows GFPuv fluorescence from transfected Sf9 cells in a 6-well culture plate with lid detected by a microplate reader. The vertical axis represents the relative fluorescence intensity value. From the left of each column, it corresponds to the relative fluorescence intensity at the time of 48 hours, 72 hours, 144 hours, and 168 hours from the start of culture. FIG. 6 shows fluorescence detection of GFPuv from transfected Sf9 cells in a conventional transparent culture plate without lid and a black plate. The vertical axis represents the relative fluorescence intensity value. From the left of each column, relative to 72 hours from the start of culture on the transparent plate, 72 hours from the start of culture on the black plate, 96 hours from the start of culture on the transparent plate, and 96 hours from the start of culture on the black plate Each corresponds to the fluorescence intensity. FIG. 6 shows a comparison of protein production estimates using FSEC (shown in bar graphs) and the method of the invention (shown in line graphs). The vertical axis represents the integrated value of the elution peak range for the data from FSEC and the relative fluorescence intensity value for the data from the method of the present invention. FIG. 5 shows an evaluation of test protein expression using the method of the present invention. SF, AKH, proteins A, B, C, and D represent the results in cells transfected with the expression vectors of vectors pCGFP_SF, pCGFP_SF + AKH, proteins A, B, C, and D, respectively.

Claims (9)

A method for quantifying the amount of a target protein expressed in cultured cells, comprising the following steps:
(1) culturing a cell capable of expressing a fusion protein of a target protein and a fluorescent protein in a culture vessel;
(2) measuring the fluorescence intensity of the fusion protein expressed in the cells cultured in (1) without disrupting the cells and without transferring them outside the culture vessel; and (3) the measured fluorescence intensity. Calculate the expression level of the target protein as an index.   The method according to claim 1, wherein the measurement of the fluorescence intensity is a downward measurement.   The method according to claim 1, wherein the fluorescence intensity is measured without removing the lid of the culture vessel. The method according to claim 1, wherein the bottom area per well of the culture vessel exceeds 0.32 cm ² .   The method according to claim 1, wherein the fluorescence intensity is measured by multipoint measurement.   The method according to claim 5, wherein a region exceeding 70% of the bottom area of the region not including the container edge of the culture vessel is subjected to multipoint measurement.   The method according to claim 1, wherein the fluorescence intensity in a culture vessel containing only the culture medium and not containing cells is also measured for normalizing the fluorescence intensity.   The method of claim 1, wherein the cell is an animal cultured cell. The method according to claim 1, wherein the measurement of the fluorescence intensity is continuously performed on the same cell culture vessel.