KR970007196B1 - Process for purifying midkine expressed in a recombinant yeast - Google Patents

Abstract

The process for purifying midcaine from a recombinant yeast observed as an insoluble refractive form of midcaine includes the steps of: (a) breaking recombinant yeast cells and centrifuging the recombinant yeast cells to obtain insoluble proteins, (b) cleaning the insoluble proteins with a buffer solution containing trypton to remove soluble proteins, (c) dissolving the insoluble proteins produced from step (b) in urea and centrifuging them, (d) diluting the supernatant of step (c) in a buffer solution, (e) eluting the diluted solution with a cation exchange resin column chromatography to separate the proteins, (f) dialyzing the eluate in a buffer solution, and (f) centrifuging the dialyzed midcaine solution and separating the supernatant with a hydrophobic column chromatography.

Description

Method for Purifying Midcaine from Recombinant Yeast

1 shows an overview of a method for isolating and purifying midkine from recombinant yeast,

FIG. 2 shows the results of electrophoresis of midkine during purification.

3 shows the bioactivity of the midkine protein.

The present invention relates to a method for purifying low purity midkine expressed from recombinant yeast by genetic engineering with high purity midkine having bioactivity.

In general, bioactive factors or growth factors are known to play an important role in the regulation of cell growth and cell differentiation (Slack, J M. et al., Nature 326, 197-200 (1987); Rosa, F. et. al., Science 239, 783-785 (1988); Mutrata, M. et al., M. et al., PNAS 85, 2434-2438 (1988); Thomas, KR et al., Nature 346, 847-850 (1990); McMahon, AP et al., Cell 62, 1073-1085 (1990)), belonging to the double fibroblast growth factor, basic FGF, int-2 and Several growth factors, such as hst, are known. They have heparin binding ability and are also called heparin binding growth factor.

Recently, new heparin-binding growth factors have been reported from several groups that show structurally significant differences from these fibroblast growth factors, for example, heparin binding neurotrophic growth-asssociated molecule (HB-GAM: Merenmies). , J. et al., J. Biol. Chem. 265, 16721 (1990)), pleiotrophin (PIN: Li, YS et al., Science 250, 1690 (1990)), heparin binding nerve growth Factor (heparin binding neurotrophic factor, HBNF: Kovesdi, J. et al., Biochem. Biophys. Res. Commun. 172, 850 (1990)), and midkine (midkine, MK: Kadomatsu, K. et al., Biochem Biophys Res. Commun. 151, 1312 (1988)).

The midkine was first identified by the Katomatsu group in the early stages of differentiation induced by retinoic acid in culturing mouse embrional carcinoma cells (EC). In other words, these cells are derived from the fact that fetal umbilical cord (EC) cells remain undifferentiated under appropriately controlled conditions, and that differentiation is induced by retinoic acid (McBurney, MW et al., Nature 299, 165 (1982)). Assuming that there is a gene involved in the differentiation process, we have successfully cloned the gene expressed early in differentiation (Kadomatsu, K. et al., BBRC 151, 1312-1318 (1988)), and this gene was on the fifth day of gestation in rats. Was found throughout the fetus on days 7-9 and only in the kidney on day 15 (Kadomatsu, K. et al., J. Cell. Biol. 110, 607-616 (1990)). . Therefore, it was named midkine because it was found in the kidney of midgestion.

Tomomura et al identified three types of cDNA that differ in the 5'-untranslated region (MK1, MK2, MK3), of which MK2 is caused by retinoic acid in fetal carcinoma cells. It was found to be derived from the major midkine RNA that is induced (Tomomura, M. et al., J. Biol. Chem. 256, 10756 (1990)), and also probes for mouse cDNA such as Tsutsui et al. Human midkine cDNA was isolated from the human fetal kidney cDNA library, and the amino acid sequence of rat and human midkine was compared to show 87% similarity (Tsutsui, J. et al., BBRC 176). , 792-797 (1991).

On the other hand, Tomomura et al. Showed that heparin-binding ability of both the midkine protein secreted early in the differentiation of mouse fetal carcinoma cells and the midkine protein isolated from fibroblast L cells transformed with the mouse midkine gene. It has been reported that it is a factor involved in the regulation of cell growth and differentiation by promoting the growth of PC12 cells (Tomomura, M. et al., BBRC 171, 603-609 (l990)).

A comparison of the amino acid sequences of heparin-bound neuronal growth factor (HBNF) and midcaine showed approximately 55% similarity, and the positions of nine cysteine residues matched, suggesting similarity of the tertiary structure of the two substances. It has been shown that midcaine, like HBNF, which already has neurostimulatory activity, has similar biological activity (Kovesdi, I. et al., BBRC 172, 850-854 (1990)). In addition, similarities between heparin binding growth-related substances / playotropins and midkine sequences have been reported to be quite high (Tsutsui et al., BBRC 176 792-797 (1991), Naito et al., BBRC 183. 701-707). (1992).

Kovesdi et al. Obtained the midkine gene from the cDNA library obtained from the human brain, expressed it in E. coli, purified the midkine protein using heparin affinity column and ion exchange resin, and measured the biological activity. Reported almost the same activity as (EP-476233 (1991)). Recently, Muramatsu et al. Reported the activity after pure purification of recombinant midkine isolated from L cells with heparin affinity column and hydrophobic chromatography (Biochem. Biophys. Res. Commun. 177, 652-658 ( 1991).

As a result of the intensive studies of the present applicant, the present inventor has succeeded in mass-expressing human midkine protein in yeast by a genetic engineering method in which the human midkine gene is fused to the ubiquitin gene (Patent Application No. _____ Reference). However, when the midkine expressed in recombinant yeast is present as an inclusion body in the yeast, it is difficult to apply the existing Muramatsu purification method directly to purify it, and the purification of the midkine expressed in E. coli In the method of Kobe's method, the contaminant protein present in the cell lysate differs significantly from the yeast, and in general, it is difficult to remove endotoxin derived from E. coli.

Therefore, the present inventors have studied to purify the mid-kine expressed in high-purity mid-caine in the yeast which is expected to be widely used in the future as one of the growth factors that have neuro-promoting activity and regulate growth and differentiation. Has led to the development of a method to purify midcaine with high purity.

Accordingly, an object of the present invention is to remove cell disruption and soluble protein, to clean up insoluble precipitate, to dissolve insoluble protein using urea, and S-cepha to purify mid-caine that is expressed in yeast and formed refractors. It is to provide a purification method useful for mass purification of high purity midkine, including steps such as rose cation exchange chromatography and phenyl-sepharose hydrophobic chromatography.

Hereinafter, the present invention will be described in detail.

Purification method of the present invention for purifying the midkine from recombinant yeast expressing the midkine,

A) crushing and centrifuging the recombinant yeast cell to obtain an insoluble protein,

B) washing the insoluble protein obtained in step a) with a buffer solution containing Triton X-100 to remove soluble protein,

C) dissolving insoluble protein from step b) with urea and centrifuging,

D) diluting the supernatant obtained from step c) with a buffer solution,

E) eluting the protein by chromatographic column chromatography of the dilution solution,

F) dialysis of the eluate into a buffer solution; and

G) centrifuging the dialysed midcaine solution to hydrophobic column chromatography of the supernatant.

Cultivation of the transformed yeast cells containing the midkine gene is carried out under the conditions in which the expression of the midkine gene occurs, which depends on the type of promoter. For example, when the midkine gene is expressed in yeast under the control of the A / G promoter (see Applicant's Korean application No. ____), 10 ml of leucine is transformed into 5% glucose. After culturing for 12 hours at 30 ° C. in a deficient medium, transfer to 50 ml of 5% YEPD medium and incubate for 1 hour to 1% 2% YEPD medium for about 48 hours to incubate. The yeast cells are precipitated by centrifugation using a separator. Precipitated cells are suspended in a suitable suspension buffer such as 1 mM EDTA, 5 mM beta-mercaptoethanol, 20 mM Tris buffer, pH 8.0. In this case, protease inhibitors such as PMSF may be added to inhibit the action of the protease, and the cell disruption process is also performed at low temperature, preferably 4 ° C., in order to minimize the degradation of the protein. Cell disruption methods include those known in the art, such as physical methods (eg, cell breakers, sonication, etc.) and non-physical methods (eg, heat treatment, freeze-thaw, osmotic shock, cytolytic enzymes). Either of these may be used, for example, cells are crushed three times for 5 minutes using a bead bead, followed by centrifugation to take a precipitate. The insoluble protein precipitate obtained above is washed with Triton X-100 solution, preferably 0.1-1% of Triton X-100 solution. At this time, it may be washed with Triton X-100 and then with 2M urea solution. Then, add urea solution and stir at room temperature for 3 hours to dissolve insoluble protein. At this time, the urea concentration is preferably 8M, and guanidine salt, sodium dodecyl sulfate, etc. may be used instead of urea, but urea is neutral There is an advantage in that exchange chromatography can be performed.

Then, the protein lysate is centrifuged by centrifugation to take a supernatant and then diluted with Tris buffer solution. As the Tris buffer solution used, 50 mM Tris buffer solution is suitable and 2M to 5M urea, preferably 2M urea. After diluting to a concentration, the supernatant was flowed to a cation exchange resin, preferably S-sepharose cation exchange resin column, which had been previously equilibrated with the same Tris buffer solution of the same urea concentration, to bind the midkine, and then to the free state. The remaining material is washed with equilibration buffer, the bound protein is washed with equilibration buffer solution containing 0.5M sodium chloride and the midkine is eluted with equilibration buffer solution containing 1M sodium chloride.

The eluted fractions were electrophoresed to identify the midkine fractions, and then the midcaine fractions were collected and dialyzed with sodium phosphate buffer solution. Preference is given to dialysis of the solution. After completion of dialysis, the supernatant is taken only by centrifugation, and the supernatant is subjected to hydrophobic column chromatography. At this time, a phenyl-sepharose column is preferable. When passing through a hydrophobic column equilibrated with an appropriate buffer in advance, the fractions which are free without binding can be collected and polyacrylamide gel electrophoresed to obtain high purity midcaine.

Activity measurement of the recombinant midkine purified according to the present invention can be carried out using the method of the above-mentioned Muramatsu (Muramatsu) using a 3T3 cell line (ATCC CCL92) can confirm the activity of the midkine.

Hereinafter, the invention will be described in detail with reference to the following Examples. The following examples are intended to illustrate the invention but do not limit the scope of the invention.

Example

Step 1. Yeast Cell Culture

Transformed yeast Saccharomyces cerevisiae DC04-UB-MK, pYLBC-A / G-UB-MK (Accession No .: ATCC 74259) containing the midkine gene, 10 ml of leucine with 5% glucose After culturing in shaken medium for 12 hours at 30 ° C., transfer to 50 ml of YEPD medium for 8 hours, incubate for 1 hour in 1L 2% YEPD medium, and incubate for about 48 hours to complete the culture when the OD reaches 20 at 650 nm. The yeast cells were precipitated by centrifugation at 3500 rpm for 25 minutes using a separator (Beckman, JB6, Rotor: JS4.2, USA) and the precipitated cells were collected.

Step 2. Cell disruption and insoluble protein isolation

5 g of the obtained midkine-expressed yeast cells were suspended in 30 ml of cell disruption buffer solution (1 mM EDTA, 5 mM beta mercaptoethanol, 20 mM Tris buffer solution (pH 8.0), and then PMSF was added to a concentration of 1 mM. Cells were crushed three times for 5 minutes using a cell crusher (Badman, Biospec Products, USA) at 10 ° C. 10 ml of the cell lysis buffer solution was added to the crushed solution, followed by stirring for 10 minutes, followed by centrifugation (Beckman, J2). -21, Rotor: JA14, USA) After centrifugation at 12000 rpm for 20 minutes, the supernatant was discarded and 40 ml of cell disruption buffer solution containing 1% Triton X-100 was added to the precipitate, followed by stirring for 10 minutes, followed by centrifugation. (12000 rpm, 20 minutes), the supernatant was discarded and the precipitate was collected.

Step 3. Dissolution of Insoluble Midcaine Protein

15 ml of 8M urea solution was added to the precipitate obtained in step 2 and stirred at room temperature for 3 hours, followed by centrifugation (Beckman, J2-21, Rotor: JA14, USA) at 12000 rpm for 15 minutes to remove insoluble materials. The supernatant in which midkine was dissolved was taken. 50 ml of 50 mM Tris buffer solution (pH 8.0) was added to the supernatant to a concentration of 2M urea, followed by centrifugation to obtain a supernatant.

Step 4. Cation Exchange Resin Column Chromatography Process

The supernatant obtained in the above example was passed through an S-sepharose cation exchange resin column (Pharmacica, Sweden, 2.5 × 10 cm), previously equilibrated with 2M urea and 50 mM Tris buffer (pH 8.0), at a rate of 10 ml / cm / hr to give a midkine. Were combined, and the materials remaining free in the column were washed with 2M urea and 50 mM Tris equilibration buffer (pH 8.0). The protein bound to the resin was sufficiently washed with the above equilibration buffer containing 0.5 M sodium chloride, eluted with the equilibration buffer containing 1 M sodium chloride, and the eluted fraction was collected by polyacrylamide gel electrophoresis to collect only the midkine fraction.

Step 5. Dialysis Process

To apply the midkine fraction obtained in step 4 to the next step, the phenyl sepharose column, three times in 50 mM sodium phosphate (pH 6.8) buffer containing 3M sodium chloride using a dialysis membrane (Spectrum, USA) of MWCO 6000-8000. Dialysis completely removed urea.

Step 6. Phenyl Sepharose Column Chromatography Process

The dialysed midcaine solution was centrifuged (12000 rpm, 30 minutes) to discard the precipitate and only the supernatant was taken and the supernatant was flowed into a phenyl-sepharose column previously equilibrated with 3M NaCl, 50 mM sodium phosphate (pH 6.8) buffer to bind. Eluted fractions were taken and polyacrylamide gel electrophoresis resulted in high purity midcaine (see FIG. 2).

In Figure 2, column 1 is the standard molecular weight protein, column 2 is the total homogenate after cell disruption, column 3 is the precipitate centrifuged in the total homogenate, column 4 is the protein dissolved in 8M urea, and column 5 is The solution was collected after S-safarose cation exchange chromatography and column 6 is the final purified midkine protein after phenyl sepharose chromatography.

Reference example. Determination of Biological Activity of Recombinant Midkine Proteins.

Activity of the recombinant midkine derived yeast purified according to the present invention was carried out using the method of Muratatsu using the 3T3 cell line (ATCC CCL92) as follows.

After the 3T3 cell line (ATCC CCL92) purchased from ATCC was finally contained in DMEM culture (Gibo-BRL. Cat. NO. 430-2100EB), 10% fetal calf serum (Sigma Cat. NO. F-2138) The cells were adjusted and laid in a 24-well culture vessel (Falcon. Cat. NO. F3047) to be ³ × 10 ⁴ cells per well, and then cultured for 24 hours under culture conditions at 37 ° C. 7% CO ₂ . Next, the supernatant was removed, and the culture medium (Gibco Cat. No. 430-2400 EB) mixed with DMEM and Ham (F-12) in a ratio of 1: 1, 10 μg / ml of insulin, and 10 μm / ml A mixed culture solution containing transferrin, 3 × 10 ⁻⁸ M sodium selenite, and 160 ng to 2.5 ng of midkine was added to each ball and incubated for 7 hours at the above culture conditions. Add ² u Ci of ³ H-thymidine (Amersham Cat. NO. TRK 686, 84 Ci / mmol) to each well, and incubate for another 16 hours, and culture the cells using a 1 ml pipette. After removal from the bottom of the vessel, ³ H-cymidine incorporated into cells was adsorbed onto a glass fiber filter mounted on a 12 well harvester (Millipore Cat. NO.XX2702550) between Millipores and other phosphorus. Uncorporated ³ H-cymidine was removed. Under the apparatus, the glass fiber filter was washed once with 1 ml of distilled water, and then washed once with 1 ml of methanol and left to dry at 60 ° C. for 30 minutes. 2 ml of scintillation cocktail (Packard Cat, No, 6013324) was added to the vial, and the amount of isotope emitted was measured by a scintillation counter (Packard Liqrid Scintillation Analyzer, 1990 CA). This result is shown in FIG. According to the present activity measurement method, the midcaine purified by the present invention shows the same activity as that of midcaine, which is known by Muramatsu et al.

Claims (11)

In the method for purifying the midkine from recombinant yeast in which the midkine is expressed in the form of an insoluble refractor, a) the step of crushing and centrifuging the recombinant yeast cells to obtain insoluble protein, b) from step a) Washing the obtained insoluble protein with a buffer containing Triton X-100 to remove the soluble protein, c) dissolving the insoluble protein from step b) with urea and centrifuging, d) Diluting the supernatant obtained from step c) with a buffer solution, e) eluting the protein by cation exchange resin column chromatography of the dilute solution, f) dialysis of the buffer solution with a dialysis solution, and g) dialysis. Hydrophobic column chromatography of the supernatant by centrifugation of the prepared midkine solution. The method according to claim 1, wherein in step b), a buffer solution of pH 8.0 containing 0.1 to 1% Triton X-100, 1 mM EDTA, 5 mM beta mercaptoethanol and 20 mM Tris is used. The method of claim 1, further comprising washing with 2M urea solution in step b). The method of claim 1 wherein said step dissolves with 8M urea in step c). The method of claim 1, wherein the 50 mM Tris buffer solution of pH 8.0 is diluted in step d). The method of claim 1, wherein dilution to 2M to 5M urea concentration in step d). The method of claim 6, wherein the dilution to 2M urea concentration in step d). The method according to claim 1, wherein S-sepharose cation exchange resin is used in step e). The method of claim 1, wherein the step e) elutes with an equilibration buffer comprising 1 M sodium chloride or 0.5 M-1.0 M concentration gradient sodium chloride, 2 M urea and 50 mM Tris. The method of claim 1, wherein the dialysis step is performed in 50 mM sodium phosphate buffer at pH 6.8. The method according to claim 1, wherein in step g), a phenyl sepharose column is equilibrated with a buffer solution of pH 6.8 containing 2 to 3 M NaCl and 50 mM sodium phosphate.