KR101542656B1 - Method for protein quantification using differential scanning fluorimetry - Google Patents

Abstract

The present invention relates to a protein quantification method using differential scanning fluorimetry, a quality control (QC) method for producing a recombinant protein using the same, and a screening method for cells overexpressing a specific protein.
The method of quantifying protein using DSF according to the present invention does not require the labeling of target protein or antibody and can quantify the protein with a small amount of sample. Especially, the protein can be directly produced from cell lysate without a separate purification process A commercially available RT-PCR instrument and plate capable of quantifying protein can be used, and 96 to 384 samples can be analyzed at the same time. Therefore, it can be quickly and easily confirmed whether the cell normally expresses the protein from the cell lysate collected from the cell incubator, and thus it can be usefully used as a means for quality control in the production process of the recombinant protein. In addition, the present invention can be applied to a method of screening cell line screening for over-expressing a specific protein for mass production of protein.

Description

[0001] The present invention relates to a protein quantification method using differential scanning fluorescence measurement,

The present invention relates to a protein quantification method using differential scanning fluorimetry, a quality control (QC) method for producing a recombinant protein using the same, and a screening method for cells overexpressing a specific protein.

Recently, interest in the use of recombinant proteins in the field of life sciences has been increasing for academic research as well as in the fields of agriculture and medicine. Currently available recombinant DNA techniques include a variety of host systems including phage, bacteria, yeast, fungi, plants, insects and mammalian cells. The choice of host system for such recombinant protein expression generally depends on the specific needs of the target protein, such as excretion, glycosylation and application fields. However, E. coli is the first choice as a heterologous protein expression system. The most important goal of heterologous protein expression in E. coli is to obtain water-soluble and active proteins with maximized yields.

Therefore, it is very important to directly or indirectly quantify the target cell to determine the state of the target cell expressed in the main cell line. Methods for detecting conventional recombinant proteins are Western blot analysis and enzyme linked immunosorbent assay. These methods require high levels of technology and relatively long experimental times as well as target protein-specific antibody or protein tags. In addition, it is impossible to directly quantitatively detect recombinant proteins from cell lysates. Accordingly, various methods for directly analyzing recombinant proteins in cell lysates have been developed.

As an example of a method for quantifying a recombinant protein in the prior art, there is a method of labeling a labeled protein such as GFP in order to easily detect expression of a recombinant protein. Although the GFP-label is a powerful tool for confirming the expression of recombinant proteins, the expressed protein must be physically modified for GFP addition so that if the original form of the protein is required, the combined GFP- There is a disadvantage in that it requires a process of removing it. The relatively large size of GFP also affects the folding and function of recombinant proteins.

Another method is a method using an anti-His tag molecular beacon aptamer (HMBA) recently reported by Xiaohong et al. Recombinant proteins fused to the His-tag can be measured directly by interaction between the His-Tag and HMBA. When bound to a heath-tagged protein, the stem portion of the HMBA is physically separated from the quencher and reorganized to emit a fluorescent signal by light irradiation. Thus, although the HMBA method is strong enough to detect a recombinant protein, it has disadvantages such that it requires both a heath-tagged recombinant protein and HMBA.

On the other hand, the melting temperature (T _m ) of a protein is defined as the temperature at which the natural and denatured proteins are equally present in equilibrium. This is an important characteristic of proteins that rely entirely on folding and unfolding conditions. The T _m can be measured using circular dichroism spectrometry (CD), differential scanning calorimetry (DSC), differential static light scatter (DSLS) and differential scanning fluorimetry (DSF) ). ≪ / RTI > DSF is a fast and cost-effective method used to study protein-ligand binding and protein stability under a variety of conditions. Using DSF, protein loosening can be monitored by increasing the fluorescence intensity of certain dyes with improved binding to the hydrophobic portion of the protein exposed during the loosening process. This can be easily performed using a commercial RT-PCR (real-time PCR) instrument.

Accordingly, the present inventors have made intensive researches to find a method for rapidly quantifying the amount of protein expressed from cells in the production of a recombinant protein. As a result, it has been found that the use of DSF, which is conventionally used for evaluating the stability of a protein, It is possible to quantify proteins directly produced from the cell lysate without a separate purification process. Thus, the present invention has been completed.

It is an object of the present invention to provide a protein quantification method using differential scanning fluorimetry, a quality control (QC) method of a recombinant protein production process using the same, and a screening method of a cell overexpressing a specific protein .

In order to accomplish the above object, the present invention provides a protein quantification method using differential scanning fluorimetry (DSF), comprising: (a) a target protein; and a dye having an improved binding force to a hydrophobic part of the protein Preparing a sample solution containing the sample solution; (b) measuring fluorescence intensity from the sample solution at a fluorescence wavelength of the dye while changing the temperature in a range of 20 to 100 캜; And (c) deriving a difference between a maximum value and a minimum value of the measured fluorescence intensity.

Preferably, the quantification is performed by comparing the protein to be quantified with a standard curve prepared from a measurement for a sample containing at least two different known concentrations.

Preferably, the protein is a purified protein or a protein contained in a cell lysate.

Preferably, the cell overexpresses a specific protein.

Preferably, the specific protein is a foreign introduced protein.

Preferably, the quantification may be a method of measuring an error range of 0 to 10% as compared with a concentration measured by enzyme activity.

Preferably, the quantification may be a method characterized by measuring directly from the cell lysate without purification of the protein to be analyzed.

In another embodiment, the present invention provides a method for producing a recombinant protein, comprising: (a) culturing a cell to express a recombinant protein; (b) collecting a sample including cells from the cell culture of step (a); (c) measuring the differential scanning fluorescence (DSF) of the cell lysate in which the collected sample is dissolved; And (d) quantifying the recombinant protein in the cell lysate from the measured DSF.

Preferably, the cell is a method characterized by being engineered to over-express a particular protein.

Preferably, the steps (b) to (d) are repeated one to ten times during the entire cell culture period.

Preferably, the quality control is a method characterized in that when the quantitative value of the recombinant protein is less than a predetermined standard in the step (d), the recombinant protein production capacity of the cell is determined to be poor considering the culture period until the measurement time.

In another aspect, the present invention provides a method for producing a cell, comprising: (a) culturing a cell to be analyzed; (b) collecting a sample including cells from the cell culture; (c) measuring the differential scanning fluorescence (DSF) of the cell lysate in which the collected sample is dissolved; And (d) determining whether or not a T _m value (melting point) exists from the DSF curve. The present invention also provides a method of screening cells overexpressing a specific protein using DSF.

Preferably from the fourth step may be a method which further comprises the step of selecting a candidate group of the protein over-expression from the T _m values for the samples determined that the T _m value is present.

Preferably, the method further comprises the step of further evaluating the activity of the selected protein candidate group to identify the protein.

According to one embodiment of the present invention, a protein quantification method using differential scanning fluorimetry (DSF) can be carried out by using a reagent and a device for measuring a conventional protein denaturation without the labeling of a target protein or an antibody specific thereto Protein quantification can be done.

According to another embodiment of the present invention, the protein quantitation method using the DSF can quantify the target protein directly from the cell lysate without producing a separate purification step in the production of the recombinant protein. Therefore, quality control of the recombinant protein production process QC).

According to another embodiment of the present invention, the protein quantitation method using the DSF can quantify the target protein directly from the cell lysate without a separate purification process, and therefore, the presence of the T _m value (melting temperature) And the cells overexpressing the specific protein can be screened.

1 is a diagram showing a melting curve for? -Amylase according to an embodiment of the present invention.
2 shows a composite plot of? (Fluorescence intensity) versus protein concentration of? -Amylase according to an embodiment of the present invention.
FIG. 3 is a graph showing a melting curve for cellobiase according to an embodiment of the present invention. FIG.
4 is a graph showing a composite plot of? (Fluorescence intensity) versus protein concentration of cellobiase according to an embodiment of the present invention.
Figure 5 is a graph showing the melting curve for E. coli MC1061 (red line) containing pHCXHD and E. coli MC1061 (blue line) containing pHCDGAS for cell lysate according to an embodiment of the present invention.
FIG. 6 is a graph showing the melting curve of E. coli DH10B (red line) containing pHis119 and E. coli DH10B (blue line) containing pHisThMA to cell lysate according to an embodiment of the present invention.
7 is a graph showing a melting curve for DGAS according to an embodiment of the present invention.
FIG. 8 is a graph showing a composite plot of? (Fluorescence intensity) versus protein concentration of DGAS according to an embodiment of the present invention.
9 is a graph showing a melting curve for ThMA according to an embodiment of the present invention.
FIG. 10 is a graph showing a composite plot of? (Fluorescence intensity) versus protein concentration of ThMA according to an embodiment of the present invention.

In order to accomplish the above object, the present invention provides a protein quantification method using differential scanning fluorimetry (DSF), comprising: (a) a target protein; and a dye having an improved binding force to a hydrophobic part of the protein Preparing a sample solution containing the sample solution; (b) measuring fluorescence intensity from the sample solution at a fluorescence wavelength of the dye while changing the temperature in a range of 20 to 100 캜; And (c) deriving a difference between a maximum value and a minimum value of the measured fluorescence intensity.

The term " differential scanning fluorimetry (DSF) "in the present invention is one of the thermal-denaturation assays for measuring the thermal stability and ligand binding of target proteins, ), Fluorescence thermal shift assay or fluorescence-based thermal shift assay. Specifically, this is carried out by introducing a dye having a binding force to a hydrophobic part of the target protein, for example, a part located inside the protein in the folded state, and measuring the fluorescence of the dye while increasing the temperature of the protein bound to the dye. Generally, as the temperature increases, the structure changes from the original folded state to the loosened state. As the structure of the protein is converted into the loosened state, the dye bonded to the inside of the protein, that is, the hydrophobic part is exposed and the fluorescence intensity thereof is increased. The switching from the folded state to the unfastened state occurs rapidly within a range of several degrees centigrade. At this time, the temperature at which the folded state and the relaxed state are equilibrated is called the melting point (T _m ) of the protein. The melting point is one of the inherent physical properties of proteins, and each protein has its own melting point value. The melting point is a temperature at which the first derivative value of the fluorescence intensity curve is maximum and the second derivative value is zero.

Most of the proteins present in nature have melting points in the range of 20 to 100 캜. Therefore, the measurement of the fluorescence intensity in the step (b) is preferably performed in the range of 20 to 100 ° C.

On the other hand, the measured fluorescence intensity may show different background intensity depending on various conditions such as temperature, pH, salt and the like. Therefore, it is preferable to perform the quantification using the difference value obtained by subtracting the minimum value from the maximum value of the fluorescence intensity depending on the fluorescence intensity of the pure dye, i.e., the background intensity, in order to correct the background intensity change.

Measurement of ligand binding to proteins using the DSF is based on the fact that binding of a specific ligand to a protein generally increases the melting point of the protein compared to the protein to which the ligand is not bound. It begins with the concept that binding of low molecular weight ligands can cause conformational changes in proteins, which can lead to changes in the thermal stability of proteins. At this time, the thermal stability change can be measured by thermally induced protein denaturation.

In an experimental aspect, the DSF can be performed by mixing a sample and a dye in a container, and then detecting the fluorescent intensity at a specific wavelength, for example, the fluorescence emission wavelength of the dye, while increasing the temperature, and plotting against the temperature. For example, it may be a purified target protein solution or a cell lysate containing the target protein, and the dye is preferably a dye having a high binding force to the hydrophobic part of the protein. Non-limiting examples of such dyes include Sypro ruby, Sypro orange, Sypro tengerine, and Sypro red. The dyes have an intrinsic absorption spectrum in the ultraviolet to visible range and have an intrinsic fluorescence spectrum in the visible range. The maximum absorption and fluorescence wavelength of these dyes depend on the type of dye, and it is therefore preferable to select absorption and fluorescence wavelengths depending on the type of dye used. For example, Sypro ruby has an absorption peak at 280 and 450 nm with a spectrum having a fluorescence peak at 610 nm, so it can excite a wavelength in the range of 430 to 470 nm and detect fluorescence at wavelengths in the range of 600 to 620 nm. Likewise, Sypro orange has a maximum absorption of 470 nm and a maximum fluorescence wavelength of 570 nm, so it can be excited to a wavelength in the range of 450 to 490 nm and fluorescence can be detected at a wavelength in the range of 560 to 580 nm. Sypro tengerine has a maximum absorption of 490 nm and a maximum fluorescence wavelength of 640 nm, so it can excite it to wavelengths in the range of 470 to 510 nm and detect fluorescence in the wavelength range of 610 to 630 nm. Also, Sypro red has a maximum absorption of 550 nm and a maximum fluorescence wavelength of 630 nm, so it can excite a wavelength in the range of 530 to 570 nm and detect fluorescence in the wavelength range of 620 to 640 nm.

Preferably, the DSF can be performed using a fluorescence spectrometer with a temperature controller, or a similar commercialized instrument, RT-PCR instrument. At this time, the temperature can be changed at a rate of 0.1 to 10 ° C / minute, preferably at a rate of 1 ° C / minute, but is not limited thereto. An accurate melting curve can not be obtained when measuring at a speed out of the above range, so that the melting point value derived from the melting curve measured at this rate is difficult to be relied upon.

The term "cell lysate" in the present invention refers to a fluid containing contents of lysed cells, and the lysis refers to the process of destroying cells The dissolution may be caused by viruses, enzymes or osmotic pressure. In the present invention, a cell lysate can be obtained by using a dissolution method known in the art without limitation as long as the cellular inclusion containing the protein is not degraded but retains its inherent characteristics. Preferably, a cell culture product containing cells and a culture medium is collected and a cell lysate is obtained by dissolving the cells by adding a known cell lysis buffer, but the present invention is not limited thereto. The cell lysis buffer is a buffer solution such as Tris-HCl for inhibiting denaturation of cell contents due to a rapid pH change, a calcium ion that is necessary for maintaining cell wall integrity, and thereby inhibits the metalloprotease activity that disrupts the cell wall and decomposes the protein Non-ionic surfactants such as SDS, dioxycholate, Triton X and / or NP-40 for separating proteins without causing denaturation of the proteins, metal chelates such as EDTA and EGTA, or zwitter ionic surfactant, or may contain a high concentration of salts such as NaCl to dissolve cells by osmotic pressure, but are not limited thereto.

Specifically, the quantification of proteins using the DSF according to an embodiment of the present invention is performed by measuring the difference between the maximum fluorescence intensity and the background fluorescence intensity, that is,? (Fluorescence intensity) in the measured melting curve given by the fluorescence intensity according to temperature, Based on the fact that it is proportional to the concentration of the protein, the concentration of the protein can be deduced by calculating Δ (fluorescence intensity) from the measured melting curve.

Preferably, the quantification may be performed by comparing the target protein to be quantified with a standard curve prepared from a measurement value of a sample containing at least two known concentrations. Since Δ (fluorescence intensity) is proportional to the concentration of the protein as described above, when plotting Δ (fluorescence intensity) measured for two or more different known protein samples at different concentrations, these plots have a linear proportional relationship The least squares method can be used to obtain an equation for a straight line for the optimized concentration versus Δ (fluorescence intensity). Further, the concentration of protein in the sample can be calculated by substituting the value of? (Fluorescence intensity) measured from the sample containing the same protein as the unknown concentration into the above equation.

The quantification method according to an embodiment of the present invention is characterized in that not only the purified protein but also the protein contained in the cell lysate can be quantified. Preferably, the cell may be a cell that overexpresses a particular protein either by intrinsic or extrinsic introduction.

Because cell lysates contain not only target proteins to be quantified but also cell contents of various components with different melting points, unlike those for proteins purified by interference by these, dissolution of widely diffused forms A curve can be obtained. However, lysates of cells that specifically express a particular protein contain the protein at a significantly higher concentration than other cell entities, and thus may have peaks similar to those in the purified protein at temperatures corresponding to the melting point of the protein . However, the quantitative method according to one embodiment of the present invention does not utilize the absolute fluorescence intensity measured, but the maximum fluorescence intensity in the melting curve Since the difference in intensity of background fluorescence is removed from the intensity, the increase effect of the background intensity due to the unspecified cell inclusion can be offset.

Further, the quantitation of protein using DSF according to an embodiment of the present invention is characterized by an error range of 0 to 10% compared with the concentration measured by enzyme activity.

In a specific embodiment of the present invention, DSF analysis of the lysates of recombinant E. coli cells containing DGAS and ThMA overexpressing vectors showed that the lysates of cells not containing the expression vector (Fig. 2 (FIG. 2), except that the background intensity is high, unlike in the case of the red dotted line (FIG. 2), which shows a sharp increase in the fluorescence intensity at a certain temperature And the temperature at which the rapid increase in fluorescence intensity was observed was found to coincide with the melting point of DGAS and ThMA measured separately. The protein concentration obtained by quantifying recombinant DGAS and ThMA from the cell lysate using the DSF was 9.1% (0.11 mg / ml vs. 0.12 mg / ml) and 5.6% (0.54 mg / ml vs 0.51 mg / ml).

Therefore, the quantification of proteins using the DSF according to an embodiment of the present invention can be directly measured from a cell lysate overexpressing the protein without purification of the protein to be analyzed.

In another embodiment, the present invention provides a method for producing a recombinant protein, comprising: (a) culturing a cell to express a recombinant protein; (b) collecting a sample including cells from the cell culture of step (a); (c) measuring the differential scanning fluorescence (DSF) of the cell lysate in which the collected sample is dissolved; And (d) quantifying the recombinant protein in the cell lysate from the measured DSF.

The process for producing the recombinant protein is not limited thereto, but is a process used as a method for mass-producing a specific protein for use in medicine and the like. The cell overexpressing the protein through gene manipulation or transfection is used for a bioreactor or the like And culturing the cells in a large amount for a predetermined period, and may further include recovery and / or purification after culturing. Since the process is characterized in that the cells are cultured in a large amount, a large amount of medium and other culture additives are used. The culture conditions for promoting cell survival, proliferation and protein expression from the cells are different from those at the laboratory level Do. Even considering the volume of medium consumed in a single production, it can range from tens of liters to thousands of liters depending on the production scale, and the cultivation period can be from days to tens of days. Particularly, it is a separate problem whether the expression of a desired protein is normally performed even if the cell grows without apparent contamination or other abnormal symptoms. Therefore, carrying out the process without a quality control process in the middle of culture may result in great loss in terms of time and / or cost. Therefore, it is important to collect the cell culture at regular intervals throughout the entire process to check whether the process is performed properly.

In addition, exhibiting an abnormally low protein expression ratio as compared with the culture day causes a decrease in productivity, while an abnormally high expression may inhibit cell growth or lower the quality of expressed protein. Accordingly, the cell culture can be periodically collected during the production process to check the cell growth rate as well as the concentration of the expressed recombinant protein, thereby regulating the growth environment. That is, when the protein expression rate is abnormally low, the culture conditions such as medium, additives, temperature and dissolved gas concentration may be appropriately adjusted, or the culture may be stopped to reduce unnecessary time and cost. Or when excessive expression proceeds, it is possible to make a judgment such as adjusting the amount of the feed or the additive, or early recovery. In this way, it is important to confirm the expression level of the protein in the middle of the process in the recombinant protein production process.

However, a method for quantifying existing protein expression requires a step of using an antibody specific to the protein or a step of marking a separate labeling substance, which is costly and time-consuming, . Since the protein assay using the DSF according to an embodiment of the present invention can be applied directly to a cell lysate obtained by dissolving a cell culture collected from an incubator, it can be analyzed quickly and easily at a low cost, and a sample of about one hundred to several hundred Can be processed at the same time. For the purpose of recombinant protein production, the cell is preferably a cell engineered to overexpress a specific protein. This is why it is possible to quantify directly from the cell lysate as described above.

In general, since the cell culture for the recombinant protein proceeds over several days to several tens of days, the steps (b) to (d) are preferably repeated one to ten times during the entire cell culture period. That is, the measurement interval and the number of times may be determined according to the type of cell, expression protein, culture medium, total cell culture period, etc., It can be adjusted flexibly according to the previous measurement result.

As described above, in particular, the quality control can be judged as having poor recombinant protein production capacity when the quantitative value of the recombinant protein is less than a predetermined standard in the step (d) considering the culture period until the measurement time.

In another aspect, the present invention provides a method for producing a cell, comprising: (a) culturing a cell to be analyzed; (b) collecting a sample including cells from the cell culture; (c) measuring the differential scanning fluorescence (DSF) of the cell lysate in which the collected sample is dissolved; And (d) determining whether or not a T _m value (melting point) exists from the DSF curve. The present invention also provides a method of screening cells overexpressing a specific protein using DSF.

In order to mass-produce the protein, it is possible to search for a cell expressing a specific protein at a high level intrinsically, or to express the gene that is expected to improve the expression by exogenously introducing it, And so on.

In this effort, a process of screening cells overexpressing a desired protein, i.e., a screening process, is required by measuring the amount of protein expressed from a candidate cell group that is expected to overexpress or overexpress a specific protein.

The presence of the T _m value in the melting curve created by the DSF for cell lysate containing other cell inclusions other than the purified protein as described above allows for the expression of a specific protein that is expressed at a relatively higher level than other cell inclusions It implies existence. Therefore, it is possible to select a cell group overexpressing a specific protein primarily by confirming the presence of the T _m value.

Furthermore, the second the protein over-expression in a cell engineered to overexpress a specific protein, the target protein by adding the step of selecting a candidate group of the protein over-expression from the T _m values for the samples determined that the T _m value is present from stage 4 , Or may identify an unknown protein that is overexpressed by intrinsic or extrinsic introduction. Since the T _m value calculated from the melting curve is a unique characteristic of a protein, a protein group having a similar T _m value can be searched and set as a protein candidate group that can be determined to be overexpressed in the system. Alternatively, in the case of a sample prepared to overexpress a known protein, it can be judged by checking whether the measured T _m value coincides with the T _m value of the corresponding protein.

Preferably, the over expressed protein can be specifically identified by further performing an activity evaluation on the selected protein candidate group. The activity evaluation can be performed using any method known in the art without limitation as appropriate to the candidate protein.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are for further illustrating the present invention, and the scope of the present invention is not limited to these examples.

Example  1: substance

(Sigma-Aldrich Chemical Co., St. Louis, Mo., USA) was added to the reaction mixture at a temperature of < RTI ID = 0.0 >Lt; / RTI > From Life Technologies (Carlsbad, CA, USA ) SYPRO Orange dye (orange dye; 5,000 × concentrated in DMSO), MicroAmp ^® Optical 96-well reaction plate quickly (Applied Biosystems, Foster City, CA , USA) and MicroAmp ^® Optical adhesive film (Applied Biosystems) was used for DSF (differential scanning fluorimetry) analysis. All other compounds were reagent grade and were purchased from Sigma-Aldrich Chemical Company and Duchefa Biochemie BV (Haarlem, The Netherlands).

Example  2: Fluorescence measurement

The complement unfolding point of the protein was measured by DSF. Samples were prepared by mixing 10 μl of SYPRO orange (10 ×) and 10 μl of enzyme solution or E. coli cell lysate (10 ×). The protein and SYPRO orange mixture was incubated in a real-time PCR (RT-PCR) system using a temperature gradient increasing from 1O < 0 > C to 99 < 0 > C. All fluorescence measurements were performed using an Applied Biosystems 7500 Fast RT-PCR system (Applied Biosystems). Sigma Plot 12.0 software (Systat Software, San Jose, USA) and Microsoft Excel program (Redmond, WA, USA) were used for all graph analysis and data statistical processing.

Example  3: E. coli Expression and purification of recombinant proteins

In order to determine the efficiency of the DSF method in quantifying the recombinant protein expression in E. coli , amylosucrase (DGAS) expressed from Deinococcus geothermalis DSM 11300 as a model protein ) And Thermus sp. ( Thermus sp . (Maltogenic amylase (ThMA)) expressed in the culture medium was used. Constitutive DGAS and ThMA expression vectors pHCDGAS and pHisThMA were constructed with a known structure. Recombinant containing ^{pHCDGAS E. coli MC1061 [F - araD139} Δ (ara-leu) 7696 gal E15 gal K16 Δ (lac) X74 rps L (Str r) had R2 (r k -m k +) mrc A mrc B1] Cells and recombinant E. coli DH10B containing pHisThMA [F ^- Φ80 lac Z M15 ( lac ZYA- arg F) U169 rec A1 end A1 hsd R17 (r _k ⁺ , m _k ⁺ ) pho sup sup E44 thi -1 gyr A96 rel A1? ^- ] cells were cultured in 100 ml of LB broth at 37 ° C for 24 hours with stirring at 200 rpm. The cells were harvested by centrifugation (7,000 x g, 4 ° C for 20 min) and washed with binding buffer containing 20 mM Tris-HCl, 500 mM NaCl and 20 mM imidazole (pH 7.0). The bacterial pellet was resuspended in binding buffer and sonicated in sonication (Sonifier 450, Branson, Danbury, CT, USA; output 4, 6x10 sec, constant duty) in an ice bath. The supernatant obtained by centrifugation (10,000 x g, 10 min at 4 [deg.] C) was used as starting material to obtain recombinant proteins and cell lysate samples.

Purification of recombinant DGAS and ThMA was performed using a fast performance liquid chromatography (FPLC) system with a HisTrap HP column (GE Healthcare, Uppsala, Sweden). The cell lysate was injected into HisTrap HP column and eluted with elution buffer (20 mM Tris-HCl, 500 mM NaCl and 500 mM imidazole (pH 7.0)) at a flow rate of 1.0 ml / min. All fractions were collected and identified by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) using 10% (w / v) acrylamide gel. Protein concentrations were determined using a BCA ™ protein assay kit (Pierce Biotechnology Inc, Rockford, IL, USA) containing bovine serum albumin (BSA) as standard.

Example  4: DGAS  And ThMA Activity analysis for

DGAS activity was determined by determining the hydrolytic activity by the dinitrosalicylic acid (DNS) method. The reaction mixture consisted of 20 l 5% sucrose, 20 l deionized water and 50 l 100 mM Tris-HCl buffer (pH 7.0). The reaction was initiated by adding 10 [mu] l of the enzyme solution to the substrate solution and lasted for 10 minutes at 45 [deg.] C. The presence of reducing sugars in the reaction mixture was determined by adding 300 [mu] l of the DNS solution and then boiling for 5 minutes. The absorbance of the final reaction solution was measured using an ultra multifunctional microplate reader (InfinteM200, Tecan, Durham, NC, USA). The reducing sugar concentration was calculated using fructose as a standard. The unit of DGAS was defined as the amount of enzyme producing 1 μmole of fructose per minute under the conditions of the assay.

The enzyme activity of ThMA was determined using 1% maltotriose (G3) dissolved in 50 mM sodium citrate buffer (pH 6.0). ThMA (50 μl) diluted in the same buffer was reacted with 50 μl of 2% G3 for 10 minutes at 60 ° C. The reaction was stopped by boiling for 10 minutes and the release of glucose from G3 was measured using the Glucose oxidase-peroxidase (GOD-POD) method. 900 [mu] l GOD-POD solution was added and incubated at 37 [deg.] C for 5 minutes to confirm the presence of D-glucose in the reaction mixture. The absorbance of the final mixture solution was measured at 505 nm using a UV / VIS spectrophotometer (DU 730, Beckman coulter, Fullerton, Calif., USA). A unit unit of ThMA was defined as the amount of enzyme catalyzing D-glucose production of 1 μmole per minute under assay conditions.

Experimental Example  One: DSF Quantification of protein using

Two commercially available enzymes α-amylase and cellobiase were used to confirm protein quantification using DSF. First, the amylase isolated from Bacillus amyloliquefaciens had a molecular weight of about 50 kDa and an optimum temperature of 65 캜. A representative DSF graph for? -Amylase from RT-PCR instrument is shown in FIG. 1A. The melting temperature (T _m ) of the protein can be obtained from the lowest point of the first derivative plot of the DSF curve and was calculated as 64.2 ± 0.4 ° C (FIG. 1A). Was; (arbitrary units AU) The highest fluorescence intensity is 1.0 mg / ml 1.6 × 10 ⁵ arbitrary units in α- amylase concentration. Fluorescence intensity was increased with increasing protein concentration. The relationship between Δ (fluorescence intensity) and protein concentration showed an excellent correlation with the R ² value of 0.986 as shown in FIG. 1B (FIG. 1B). This suggests that the protein concentration can be measured from? (Fluorescence intensity) of the DSF graph.

The same experiment was performed with cellobiase (also called Novozyme 188) which hydrolyzes cellobiose to two glucose molecules. The cellobiase was obtained by submerged fermentation of Aspergillus niger . Although the values of the highest fluorescence intensities of the curves were not the same, the pattern of the DSF curve observed for cellobiase was consistent with that of? -Amylase. The T _m of cellobiase was calculated as 66.7 ± 0.2 ° C (Figure 1C). The highest fluorescence intensity was 2.9 × 10 ⁴ AU at a cellobiose concentration of 0.6 mg / ml. As shown in? -amylase, there was a strong correlation between? (fluorescence intensity) and the protein concentration of cellobiase, and the R ² value was 0.994 (FIG. 1D). The above results indicate that protein concentration can be analyzed with high reliability by DSF.

DSF has been used as a fast and attractive screening method to identify low molecular weight ligands that bind to proteins and promote stability. In addition, T _m was analyzed by DSF at high throughput of monoclonal antibodies in the presence of a surfactant. In DSF analysis, protein annealing can be analyzed by increasing the fluorescence of certain dyes, such as SYPRO orange, which have affinity to the hydrophobic portion of the protein. The dye has weak fluorescence in the free state of water or a hydrophilic environment, but strongly fluoresces when bound to the exposed hydrophobic region in the denatured protein. Although the actual fluorescence intensity was different, both α-amylase and cellobiase exhibited typical protein denaturation curves in DSF plots obtained from RT-PCR. The difference in fluorescence intensity can be attributed to differences in the hydrophobicity of the protein. More SYPRO oranges bind to the modified cellobiase having an internal hydrophobic than? -amylase, which exhibits higher fluorescence intensity in DSF. However, the most important feature is that Δ (fluorescence intensity) is closely related to the concentration of both proteins, suggesting that DSF can be used as a quantitative detection method for proteins in solution.

Experimental Example  2: E. coli In the melt  Of recombinant protein DSF  analysis

Cell lysates of several E. coli strains used in recombinant DNA technology were used for DSF analysis. The E. coli strains used were E. coli DH10B, MC1061 and BL21, which showed similar DSF curve patterns. The presence of a typical cloning vector such as pUC19 or pGEM3Z influenced DSF analysis. However, the higher baseline fluorescence intensity (ca. 3.0 × 10 ⁵ -4.0 × 10 ⁵ AU) compared to that observed in the DSF analysis of α-amylase and cellobiase in the buffer is due to the various components of the cell lysate can do.

Cell lysates obtained from recombinant E. coli cells were subjected to DSF analysis. E. coli containing the constant expression vector for DGAS, pHCDGAS, exhibited a different DSF curve pattern than the control E. coli showing a large increase in fluorescence intensity at around 65 DEG C (FIG. 2A). The T _m of DGAS is 59.3 ± 0.7 ° C, suggesting that increased fluorescence near 65 ° C is due to denaturation of recombinant DGAS in E. coli cell lysates. Indeed, the highest fluorescence intensity of DSF using purified DGAS was observed at the same temperature as the maximum in the DSF curve of recombinant E. coli cells containing DGAS. Despite the presence of some differences in Δ (fluorescence intensity) due to different expression levels of foreign proteins in different E. coli host systems, the pattern of the DSF graph was identical regardless of the E. coli host strain used .

In addition, E. coli DH10B cells containing pHisThMA, a constitutive expression vector for ThMA, were identified by DSF (FIG. 2B). The DSF curve showed a large protrusion (T _m = 74.5 ± 0.3 ° C) near 75 ° C, which is the T _m of ThMA. This suggests that the protrusions show denaturation of recombinant ThMA expressed in E. coli , similar to the DGAS study. The same results were obtained from E. coli MC1061 and BL21, indicating that the DSF method can be applied to most E. coli cells.

Experimental Example  3: DSF Using E. coli Quantification of recombinant proteins expressed in

Using purified DGAS as a model protein, a standard curve for the relationship between delta (fluorescence intensity) and protein concentration was obtained (Figures 3A and B). The protein concentration used ranged from 11.3 μg / ml to 0.133 mg / ml. At this time, the fluorescence intensity difference was increased with increasing protein concentration, and the coefficient of determination (R ² value) between Δ (fluorescence intensity) and protein concentration was 0.986. This indicates that there is a close relationship between the two variables. And 0.12 ± 0.01 mg / ml of DGAS expressed in the recombinant cell lysate determined using DSF. The specific activity of the purified DGAS was calculated to be 59.09 ± 2.49 U / mg. The activity of the cell lysate was 6.75 ± 0.06 U / ml, which was expressed in the recombinant cell lysate at a concentration of 0.11 ± 0.00 mg / ml DGAS. These values are very close to the concentrations obtained from the DSF analysis suggesting that direct quantification of recombinant proteins expressed in E. coli is possible by the DSF method.

The same experiment was performed with ThMA (Fig. 3C and D). The R ² value of the relationship between Δ (fluorescence intensity) and protein concentration was 0.988, which was very close to the result for DGAS. Therefore, Δ (fluorescence intensity) and protein concentration also showed a strong correlation with ThMA expression. The specific activity of purified ThMA was 24.49 ± 1.20 U / mg, the determined activity from the cell lysate was 13.16 ± 0.36 U / ml, and the concentration of ThMA expressed in the recombinant cell lysate was 0.54 ± 0.02 mg / ml. The above values were in agreement with the ThMA concentration obtained from DSF experiments of 0.51 ± 0.07 mg / ml.

As a quantitative method according to the present invention, activity and quantitative data for recombinant DGAS and ThMA protein expressed from E. coli by DSF measurement using cell lysate are summarized in Table 1 below.

As a result, it was confirmed that a specific protein can be quantified using differential scanning fluorometry (DSF), and quantification can be performed without purification of protein.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

Claims (14)

As a protein quantification method using differential scanning fluorimetry (DSF)
(a) a sample solution containing a dye having an intrinsic absorption spectrum in the ultraviolet to visible light range and having an intrinsic fluorescence spectrum in the visible light region, the protein having binding force to a hydrophobic portion of the protein by including a target protein and a hydrophobic portion; ;
(b) measuring fluorescence intensity from the sample solution at a fluorescence wavelength of the dye while changing the temperature in a range of 20 to 100 캜;
(c) deriving a difference between a maximum value and a minimum value of the measured fluorescence intensity; And
(d) comparing the value derived from step (c) with a standard curve drawn from measurements for a sample containing the target protein at two or more different known concentrations.
delete The method according to claim 1,
Wherein the protein is a purified protein or a protein contained in a cell lysate.
The method of claim 3,
Wherein the cell overexpresses a specific protein.
5. The method of claim 4,
Wherein the specific protein is a foreign introduced protein.
The method according to claim 1,
Wherein said quantification is measured within an error range of less than 10% as compared to the concentration measured by enzyme activity.
The method according to claim 1,
Wherein the quantification is directly measured from the cell lysate without purification of the protein to be analyzed.
(a) culturing a cell to express a recombinant protein;
(b) collecting a sample including cells from the cell culture of step (a);
(c) the cell lysate in which the collected sample is dissolved has a binding force to a hydrophobic part of the protein by including a hydrophobic part and has an intrinsic absorption spectrum in a range of ultraviolet to visible light, and has an intrinsic fluorescence spectrum Adding a dye having the formula: < RTI ID = 0.0 > (DSF) < / RTI >
(d) deriving a difference between a maximum value and a minimum value of fluorescence intensity from the measured DSF;
(e) quantifying the recombinant protein in the cell lysate by comparing the value derived from step (d) with a standard curve prepared from a measurement value for a sample containing the recombinant protein at two or more different known concentrations; And
(f) comparing the quantified value with a normal value to determine whether the culture condition or the amount of the additive is adjusted or whether the culture is continued if abnormally low or high, and the quality control of the recombinant protein production process ; QC) method.
9. The method of claim 8,
Wherein said cell is engineered to overexpress a specific protein.
9. The method of claim 8,
Wherein the steps (b) to (d) are repeated one to ten times during the entire cell culture period.
9. The method of claim 8,
Wherein the quality control determines that the recombinant protein production capacity of the cell is poor when the quantification value of the recombinant protein is less than a predetermined standard in the step (d) considering the culture period until the measurement time.
(a) culturing a cell to be analyzed;
(b) collecting a sample including cells from the cell culture;
(c) the cell lysate in which the collected sample is dissolved has a binding force to a hydrophobic part of the protein by including a hydrophobic part and has an intrinsic absorption spectrum in a range of ultraviolet to visible light, and has an intrinsic fluorescence spectrum Adding a dye having the formula: < RTI ID = 0.0 > (DSF) < / RTI >
(d) determining whether a T _m value (melting point) exists from the DSF curve; And
(e) determining the presence of a specific protein expressed at a relatively high level relative to other cell inclusions in the presence of the T _m value, using a DSF.
13. The method of claim 12,
The method of screening comprises from step (d) the further step of selecting a candidate group of the protein over-expression from the T _m values for the samples determined that the T _m value is present.
14. The method of claim 13,
And further evaluating the activity of the selected protein candidate group to identify the protein.

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