KR101884793B1 - Method for production and purification of CAGE protein - Google Patents

Abstract

Provided is a production method for expressing a soluble CAGE protein under specific incubation condition. Moreover, provided is a method for purifying the CAGE protein through a combination of a plurality of isolation steps. The method for producing, isolating, and purifying the CAGE protein of the present invention enables high purity isolation and purification of the CAGE protein, contributes to research of cancer-related mechanisms associated with the CAGE protein by using the same, and can be useful in fields of cancer-related research associated with the CAGE protein.

Description

≪ Desc / Clms Page number 2 > Production and purification of CAGE protein {

The present invention relates to a method for producing and purifying proteins, and more particularly, to a method for producing and purifying CAGE proteins.

Recently, there have been reports that cancer is related to the immune system. In order to study cancer-related mechanisms of CAGE (cancer-associated antigen, cancer / test antigen, DDX53) Purification is required in the related field.

The CAGE protein (cancer / testis antigen CAGE) is referred to as cancer-associated antigen or DDX53 protein and serologic analysis of recombinant cDNA library expression (SEREX) technique for cDNA expression library prepared in gastric cancer cell lines and testis tissue (Cho B., 2002, Biochem . Biophys . Res. Commun . , 295, 715-726), which is specifically present in the serum of gastric cancer patients. In the present study, we investigated whether CAGE gene methylation is related to the degree of DNA demethylation of CpG island (CpG island) and CAGE expression (Cho B., 2003, Biochem . Biophys . Res. Commun . , 307 , 52-63), suggesting the possibility of a new method of diagnosing cancer through analysis of the methylation pattern of the CAGE gene.

In addition, overexpression of the CAGE gene increases cell mobility and phosphorylation of ERK, p38 MAPK, and FAK increases cell migration by CAGE (Shim H., 2006, Mol Cells , 21, 367-375) , CAGE and CAGE-derived peptides have been reported to increase the activity of cytotoxic T lymphocytes (Shim, E., 2006, Biotechnol . Lett. 28, 515522).

However, until now, the CAGE protein has become insoluble at the time of recombinant expression, and it has been difficult to separate and purify it. Therefore, it is urgent to study the separation and purification technology of CAGE protein in order to study CAGE protein-related cancer-related mechanism.

Korean Patent Publication No. 10-2014-0037726 (Mar. Korean Patent Publication No. 10-2013-0010897 (Feb.

Schlager et al. BMC Biotechnology 2012, 12:95 Palmer et al. Curr Protoc Protein Sci. 2004 November; CHAPTER: Unit-6.3.

The inventors of the present invention have studied a method of expressing CAGE protein as a soluble protein for the isolation and purification of CAGE protein for cancer-related mechanism-related mechanism of CAGE protein. After Escherichia coli was first cultured at a general strain culture temperature, The CAGE protein can be expressed in a soluble state when isopropyl β-D-1-thiogalactopyranoside is added and cultured at a specific temperature, and then the CAGE protein is isolated and purified by a combination of multiple separation processes .

Accordingly, it is an object of the present invention to provide a production method of a specific culture condition capable of expressing a CAGE protein in a soluble state, and a method of purifying the CAGE protein by a combination of a plurality of separation processes.

According to an aspect of the present invention, there is provided a method for producing a recombinant plasmid comprising: (a) a primary culture step of culturing an Escherichia coli containing a CAGE (DDX53) gene-inserted plasmid at 35 to 38 ° C; And (b) a second culture step in which isopropyl β-D-1-thiogalactopyranoside is added to the primary cultured medium and cultured at 10 to 20 ° C.

In one embodiment, E. coli can be cultured at 37 DEG C in step (a), and cultured at 16 DEG C in step (b).

According to another aspect of the present invention, there is provided a method for producing a cucurbitidase inhibitor, comprising the steps of: (c) adding sodium lauroyl sarcosinate to a pellet obtained by centrifuging Escherichia coli containing solubilized CAGE protein; (d) sonicating and lysing the lysed cells; (e) centrifuging the disrupted cell lysate and purifying the supernatant with a Q-anion exchange column; (f) purifying the Ni ^{2 +} -charged Ni-NTA column; And (g) purifying by size exclusion chromatography.

In one embodiment, sodium lauroyl sarcosinate in step (c) may be added in an amount of 0.1 to 1 wt%, preferably 0.5 wt%.

In one embodiment, the Ni ^{2 +} -charged Ni-NTA column of step (f) can be eluted at a flow rate of 5 ml / min and the size exclusion chromatography of step (g) is performed at a flow rate of 1 ml / min It can be eluted.

In one embodiment, the CAGE protein may be 75 kDa.

According to another aspect of the present invention, there is provided a method for producing a recombinant E. coli strain comprising the steps of: (a) a primary culture step of culturing E. coli containing a plasmid into which a CAGE (DDX53) gene has been inserted at 35 to 38 占 폚; (b) a secondary culture step in which isopropyl β-D-1-thiogalactopyranoside is added to the primary cultured medium and cultured at 10 to 20 ° C; (c) adding sodium lauroyl sarcosinate to the pellet obtained by centrifuging the cultured Escherichia coli to dissolve the pellet; (d) sonicating and lysing the lysed cells; (e) centrifuging the disrupted cell lysate and purifying the supernatant with a Q-anion exchange column; (f) purifying the Ni ^{2 +} -charged Ni-NTA column; And (g) purifying by size exclusion chromatography.

According to the present invention, Escherichia coli is first cultured at a culture temperature of a general strain, and isopropyl β-D-1-thiogalactopyranoside is then added, followed by secondary culture (low temperature expression) at a specific temperature, when being observed in the CAGE protein (75 kDa) in the case of the supernatant added to the carbonate obtained by dissolving a soluble protein, a combination of a number of separate process after the (Q- anion exchange column chromatography, Ni ^{2 +} - charged Ni-NTA Column affinity chromatography, size exclusion chromatography) to separate and purify the CAGE protein.

Therefore, it has become possible to separate and purify the CAGE protein with high purity by the production, separation and purification of the CAGE protein. Therefore, it can contribute to the study of the CAGE protein-related cancer-related mechanism and is useful in the CAGE protein- Lt; / RTI >

1 is a schematic diagram of a DDX53 motif.
FIG. 2 is a photograph showing SDS-PAGE in which proteins are expressed as insoluble and contained in pellets (P) when proteins are expressed at 37 ° C and 16 ° C in an E. coli cell line.
FIG. 3 shows that the expression of the protein in the E. coli cell line was all insoluble at 37.degree. C. (A) when 0.5% Sarkosyl was added and when expressed at 16.degree. C., the supernatant (S) (B) a soluble protein.
FIG. 4 is a graph showing the results obtained by filtering soluble DDX53 protein using a Q-anion exchange column in a volume of 5 ml, and then performing affinity chromatography [Ni ²⁺ -charged Ni- NTA column] is serially connected in series to sequentially purify the DDX53 protein.
Figure 5 shows the absorbance (280 nm) of each fraction according to elution time (flow rate: 5 ml / min) separated by Ni ^{2 +} -charged Ni-NTA column (volume: 5 ml) U: Unbounded fraction, W: Washing, Protein size marker), which shows two bands (75 kDa and 20 kDa) on P3 as a result of SDS-PAGE analysis. P1: Peak 1, P2: Peak 2, P3: Peak 3, P4: Peak 4, P5: Peak 5].
FIG. 6 shows the results obtained by measuring the absorbance (280 nm) (A) and the SDS-PAGE (B) of the fraction according to the elution time (flow rate 1 ml / min) separated by size exclusion chromatography , And a migration band of 75 kDa.
FIG. 7 shows the results of confirming that the 75 kDa band obtained through size exclusion chromatography was a CAGE protein by Western blotting.

(A) a primary culture step of culturing Escherichia coli containing a plasmid into which CAGE (DDX53) gene is inserted at 35 to 38 占 폚; And (b) a secondary culture step in which isopropyl β-D-1-thiogalactopyranoside is added to the primary cultured medium and cultured at 10 to 20 ° C.

Step (a) is a primary culture step in which E. coli containing a plasmid into which the CAGE (DDX53) gene is inserted is cultured at a normal culture temperature of 35 to 38 ° C. Cultivate until A ₆₀₀ reaches 0.6. The incubation temperature is preferably 37 占 폚, but is not limited thereto.

Step (b) is a step of further culturing the primary cultured medium at a low temperature. At this time, isopropyl β-D-1-thiogalactopyranoside is added and cultured, and the CAGE protein is expressed at low temperature by culturing at a lower temperature than the primary culture.

The incubation temperature suitable for low temperature expression is 10 to 20 째 C, preferably 14 to 18 째 C, more preferably 16 째 C, but is not limited thereto. The additional incubation time is 10 to 20 hours, preferably 12 to 18 hours, more preferably 15 hours, but is not limited thereto.

The isopropyl β-D-1-thiogalactopyranoside may be added in an amount of 0.5 to 1 mM based on the total amount, preferably 1 mM of the total amount.

According to another aspect of the present invention, there is provided a method for producing a cucurbitidase inhibitor, comprising the steps of: (c) adding sodium lauroyl sarcosinate to a pellet obtained by centrifuging Escherichia coli containing solubilized CAGE protein; (d) sonicating and lysing the lysed cells; (e) centrifuging the disrupted cell lysate and purifying the supernatant with a Q-anion exchange column; (f) purifying the Ni ^{2 +} -charged Ni-NTA column; And (g) purifying by size exclusion chromatography.

Step (c) is a step of adding sodium lauroyl sarcosinate to the pellet obtained by centrifuging Escherichia coli containing the CAGE protein, which has been solubilized by the low-temperature expression step, and dissolving it.

In one embodiment, the sodium lauroyl sarcosinate may be added in an amount of 0.1 to 1% by weight, preferably 0.5% by weight.

The sodium lauroyl sarcosinate has the IUPAC designation sodium [dodecanoyl (methyl) amino] acetate (CAS number 137-16-6, C ₁₅ H ₂₈ NNaO ₃ , Molecular weight 293.38 g / mol), also referred to as sarkosyl.

Step (f) is a step of separating the His-tag-bound protein by affinity chromatography using a Ni ^{2 +} -charged Ni-NTA column and eluting at a flow rate of 5 ml / min.

Step (g) is a step of separating a protein having a specific molecular weight using size exclusion chromatography, and can be eluted at a flow rate of 1 ml / min.

In one embodiment, the CAGE protein may be 75 kDa.

According to another aspect of the present invention, there is provided a method for producing a recombinant E. coli strain comprising the steps of: (a) a primary culture step of culturing E. coli containing a plasmid into which a CAGE (DDX53) gene has been inserted at 35 to 38 占 폚; (b) a secondary culture step in which isopropyl β-D-1-thiogalactopyranoside is added to the primary cultured medium and cultured at 10 to 20 ° C; (c) adding sodium lauroyl sarcosinate to the pellet obtained by centrifuging the cultured Escherichia coli to dissolve the pellet; (d) sonicating and lysing the lysed cells; (e) centrifuging the disrupted cell lysate and purifying the supernatant with a Q-anion exchange column; (f) purifying the Ni ^{2 +} -charged Ni-NTA column; And (g) purifying by size exclusion chromatography.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the following examples are intended to illustrate the present invention, and the scope of the present invention is not limited thereto.

Example 1. E. coli in  Low Temperature Expression of CAGE Protein

(One) E. coli cells containing a plasmid containing the DDX53 (CAGE) gene were cultured in LB medium supplemented with 50 μg · ml ^-1 kanamycin (37 ° C, A ₆₀₀ = 0.6).

Specifically, the plasmid in which the DDX53 (CAGE) gene was inserted was designated as the forward primer 5'-ggg ccatgg gcatgcatcatcatcatcatacacatgtcccactgggccc-3 'and the reverse primer 5'-ggg ggatcc tcaacttaaaaaataaaactccttgcg-3' (NcoI and BamHI restriction sites are underlined respectively) Were amplified from human cDNA by polymerase chain reaction (PCR). The amplified DDX53 (CAGE) gene was then cloned into pET-28a (Novagen, Madison, Wis., USA) expression plasmid for expression of DDX53 (CAGE) having a direct 6-histidine label at its N-terminus. The nucleotide sequence of the gene to be expressed was confirmed by automatic sequencing.

E. coli BL21 (DE3) RIPL was transformed for expression of DDX53 (CAGE) protein tagged with Direct 6-histidine tag. 1 L of fresh LB (Luria-Bretani) medium containing 50 mg / ml kanamycin and containing 10 g / L of Bacto Tryptone, 5 g / L of yeast extract and 10 g / , 10 ml of BL21 (DE3) RIPL cells were added to each well and cultured at 37 DEG C until OD600 reached about 0.6.

(2) The cultured E. coli culture solution was quenched and allowed to stand at 4 DEG C for 60 minutes. 1 mM IPTG (isopropyl beta-D-1-thiogalactopyranoside) was added to induce the expression of DDX53 (CAGE) in the culture medium of E. coli. After incubation at 37 ° C for 4 hours and at 16 ° C (or 37 ° C) Cells were cultured.

(3) The cultured E. coli cells were centrifuged (5,000 rpm, 10 minutes).

(4) The supernatant was removed and the cell pellet was washed with lysis buffer (20 mM HEPES, 300 mM NaCl, 10% glycerol, and 0.5 mM β-mercaptoethanol, pH 8.0) and centrifuged again at the same rpm to obtain clean pellet .

(5) Lysis buffer (20 mM HEPES, 300 mM NaCl, 10% glycerol, and 0.5 mM [beta] -mercaptoethanol) supplemented with or without 0.5% sacosyl [sodium lauroyl sarcosinate] , pH 8.0) was added to the cell pellet to release the cells.

(6) The cells were disrupted using an ultrasonic wave crusher (amplitude: 34%, reaction time: 10 min, pulse on: 2 sec, pulse off: 2 sec).

(7) The disrupted cells were centrifuged (15,000 rpm, 20 min) to separate supernatant and pellet. The presence of CAGE protein in the supernatant and pellet was confirmed by 12.5% SDS-PAGE.

As a result, as shown in Fig. 2, when the protein was expressed at 37 DEG C and 16 DEG C without adding saccharose in the E. coli cell line, the protein (75 kDa) was expressed as insoluble in both conditions, (pellet, P).

However, as shown in Fig. 3, the solubility of the extracted protein in the condition of adding 0.5% sacocyl to the culture conditions of 16 ° C was lowered at two temperature conditions (37 ° C and 16 ° C) in which 0.5% It was confirmed that the solubility was improved than that induced (see Fig. 3). In the E. coli cell line, the protein was present in the insoluble state at 37 ° C, but when CAGE protein (75 kDa) was added at 0.5% sucrose and 16 ° C, the supernatant, S) and (B) it was confirmed to be expressed as a soluble protein.

Example 2. E. coli in  Purification of Expressed CAGE Protein

(1) Pellet was removed from E. coli ( cells expressing CAGE protein in supernatant) supplemented with 0.5% saccharose at a low temperature of 16 ° C by ultrasonic cell disruption and centrifugation, and the supernatant was purified by Q - Mixture of Ni ²⁺ -charged Ni-NTA column (volume: 5 ml) (HisTrap ^™ HP, GE Healthcare, Sweden) with anion exchange column (volume: 5 ml) (HiTrap ^™ Q HP, GE Healthcare, Sweden) (See Figure 4). ≪ / RTI >

(2) After the sample loading, the Ni-NTA column was attached to FPLC (Fast Protein Liquid Chromatography) equipment and the protein was separated / purified by imidazole gradient (step-wise) method. The eluate was separated into P1, P2, P3, P4 and P5 according to the elution time, and absorbance was measured at 280 nm. Each fraction was confirmed by SDS-PAGE (see FIG. 5). As shown in Fig. 5, a protein showing two bands (75 kDa and 20 kDa) in P3 was identified.

(3) The P2 and P3 fractions obtained above were concentrated to 500 μl using Centricon (MW: 30,000).

(4) The concentrated protein samples were centrifuged at 13,000 rpm for 10 minutes, and syringe filters (pore size: 0.20 μm) were used to aggregate or remove impurities.

(5) The protein sample from which impurities were removed was separated into a column (Superdex 200 10/300 GL column) attached to the FPLC, and absorbance was measured at 280 nm. Each fraction was confirmed by SDS-PAGE (see FIG. 6). As shown in Fig. 6, it was confirmed that two bands (75 kDa and 20 kDa) were separated.

(6) The 75 kDa band obtained by size exclusion chromatography was confirmed to be a CAGE protein by Western blotting (see FIG. 7).

Claims (10)

A first culture step of culturing E. coli containing a plasmid in which the CAGE (DDX53) gene is inserted at 35 to 38 DEG C;
(b) a secondary culture step in which isopropyl β-D-1-thiogalactopyranoside is added to the primary cultured medium and cultured at 16 ° C;
(c) adding 0.5% by weight of sodium lauroyl sarcosinate to the pellet obtained by centrifuging the cultured Escherichia coli and dissolving it;
(d) sonicating and lysing the lysed cells;
(e) centrifuging the disrupted cell lysate and purifying the supernatant with a Q-anion exchange column;
(f) purifying the Ni ²⁺ -charged Ni-NTA column; And
(g) Purification by size exclusion chromatography
≪ RTI ID = 0.0 > CAGE < / RTI > The method according to claim 1, wherein Escherichia coli is cultured at 37 DEG C in step (a). delete delete delete delete The method of claim 1, wherein the Ni ²⁺ -charged Ni-NTA column of step (f) is eluted at a flow rate of 5 ml / min. The method of claim 1, wherein said size exclusion chromatography of step (g) is eluted at a flow rate of 1 ml / min. The method of claim 1, wherein the CAGE protein is 75 kDa. delete

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