KR100373863B1 - Purification of basic peptide or protein from fusion protein - Google Patents

Abstract

The present invention relates to a method for separating a basic peptide or basic protein in high yield from a fusion protein having a hydroxylamine cleavage site between the basic peptide or basic protein and the fusion partner. Specifically, the present invention consists in reacting with hydroxyamine at pH 7.5-8.5 and recovering basic peptides from the reactants.

Description

Method of separating basic peptide or protein from fusion protein {Purification of basic peptide or protein from fusion protein}

The present invention relates to a method for effectively purifying and recovering a basic peptide or basic protein from recombinant cells.

Peptides or proteins with biological activity such as antimicrobial activity or cytotoxic activity are often basic, and in general, it is very difficult to express such a basic peptide or basic protein in high yield in recombinant cells. This is because the produced peptide is rapidly degraded in the cell, or the host cell is killed due to the cytotoxicity of the produced peptide, and the expression level is low, and it is difficult to separate from other proteins and peptides. In order to solve this problem, a method of expressing a desired peptide in the form of a fusion protein has recently been used.

However, the method of expressing in the form of a fusion protein can be expected to prevent the degradation of the resulting peptide in the cell or the death of the cell due to the cytotoxicity of the produced peptide, the desired protein or There is a problem that the yield of separating the peptide is very low.

In fusion proteins or fusion peptides, we can separate the desired peptide from the fusion protein by introducing an appropriate recognition sequence between the fusion partner and the peptide and then cleaving the desired peptide and the fusion partner in the expressed fusion protein. . Such cleavage methods include enzymatic methods using enzymes and chemical methods using chemicals.

CNBr and hydroxylamine are known as compounds used in the chemical method, but the protein yield obtained by the chemical method using these substances is very low, about 40%. Antorini et al. (Protein Expression and Purification 11,135-147, 1997) present the conditions that affect the purification of fusion proteins with hydroxylamine cleavage sites with hydroxyamines. Reaction pH 9.5 was presented, reporting high protein recovery at high pH. It was reported that IGF-I was obtained in a yield of about 50-60% by cleaving the fusion protein at a reaction pH of 9.5 and reducing the amount of fusion protein to 1-4 mg / M.

The present invention provides a method for effectively separating a desired basic peptide from a fusion protein comprising a hydroxylamine cleavage site and a basic peptide.

The present invention also provides a method for purifying the basic peptide in high yield from recombinant cells.

1 is a chromatogram showing that MSI-344 was cleaved by hydroxyamine reaction.

Figure 2 is a chromatogram showing that Buporin II was cleaved by hydroxyamine reaction.

Figure 3 is a chromatogram showing that Buporin IIb was cleaved by hydroxyamine reaction.

In the present invention, the fusion protein means a fusion protein having a hydroxylamine cleavage site between the foreign basic peptide or the basic protein and the fusion partner.

When the foreign peptide or protein to be recovered is a basic peptide or a basic protein, the present inventors remarkably improve the recovery yield of a desired peptide or protein by adjusting the reaction pH to a relatively low range of pH 7.5 to 8.5 unlike those proposed in the prior art. Found that it can be increased.

The present invention relates to a method for recovering a basic peptide or basic protein from a fusion protein.

The recovery method according to the present invention consists in recovering the basic peptide from the reactant after reacting the basic peptide or a fusion protein having a hydroxylamine cleavage site between the basic protein and the fusion partner at pH 7.5 to 8.5.

The present invention relates to a method capable of purifying peptides in high yield from recombinant cells.

Purification method according to the present invention comprises the steps of recovering the cells from the fermentation medium; Collecting the fusion protein from cell lysate or culture medium; Reacting the fusion protein with hydroxylamine at pH 7.5-8.5; And recovering the basic peptide or basic protein from the reaction.

When the fusion protein is secreted extracellularly in the method of the present invention, the fusion protein is collected from the cell culture and treated with hydroxyamine.

If the fusion protein is present in the cell in the method of the present invention, the cell is disrupted and the fusion protein is collected and treated with hydroxyamine.

In the method of the present invention, the reaction time is 10 to 24 hours, preferably 15 to 22 hours.

By this method, the basic peptide or basic protein can be recovered from the fusion protein in a yield of about 70 to 90%.

The method of the present invention is suitable for separating basic peptides or basic proteins from fusion proteins, and examples of basic peptides include merginine, buforin, defensins, derivatives thereof, and the like, and examples of basic proteins are histones. The basic peptide or basic protein according to the present invention is preferably a basic peptide or protein having a pI of 10 or more.

Gene sequence for expressing the peptide used in the present invention is as follows.

MSI-344

Sma i

GGAT CCC GGG ATC GGC AAA TTC CTG AAA AAG GCT AAG AAA TTT GGT AAG GCG TTC GTT

G I G K F L K K A K K F G K A F V

AAA ATC CTG AAA AAG TAATG AAGGA GATAT ATTAAT GGATCC

K I L K K RBS Ase I

Buporin II

GGG ACC CGT TCC TCC CGT GCT GGT CTG CAG TTC CCG GTT GGT CGT GTT CAC CGT CTG

G T R S S R A G L Q F P V G R V H R L

CTG CGT AAA TAA TGA AGG AGA TAT ATT AAT GGATCC

LRK Bam HI

Buporin IIb

GGG CGT GCT GGT CTG CAG TTC CCG GTT GGT CGC CTG CTG CGC CGT CTG CTG CGT CGC

G R A G L Q F P V G R L L R R L L R R

CTG CTG CGC TAA TGA AGG AGA TAT ATT AAT GGATCC

LLR Bam HI

In the purification method according to the present invention, it is preferable to add a protease inhibitor in order to prevent degradation by the protease present in the cells or fermentation broth. Phenylmethylsulfonyl fluoride (PMSF), ethylene diamine tetraacetic acid (EDTA), benzamidine, ZnCl _2, etc. may be used as the inhibitor. The amount of inhibitor will vary depending on the fusion protein, recombinant microorganism, type of inhibitor, and the like.

The fusion peptide or fusion protein is accumulated in cells or secreted into the culture medium depending on the type of peptide or protein to be produced, the fusion partner or cell to be used, and the like. When accumulating in cells, the cells are separated from the culture medium or the culture medium itself is crushed using a sonicator, a microfluidizer, a wheat or the like.

As a method for recovering the fusion protein from the cell lysate or the fermentation broth, the fusion protein may be recovered, for example, by centrifuging the cell lysate or the fermentation broth or through a membrane.

The recovered fusion protein is pretreated to remove impurities if necessary prior to hydroxylamine reaction. For example, cation exchange resin chromatography can be performed. In particular, when the fusion protein is insoluble, it is possible to obtain a fusion protein with increased purity by dissolving in a urea or guanidine solution capable of dissolving the fusion protein, precipitating impurities, and centrifuging to remove the precipitated impurities.

In case of recovering the insoluble material through the membrane, the pretreatment process prior to the hydroxylamine reaction can be omitted because the insoluble material recovery effect and the impurity removal effect can be obtained by adjusting the membrane material, the cut off value, and the flow rate. It is preferable.

The amount of hydroxylamine used may vary depending on the type of protein desired, the type of fusion partner, the size of the fusion protein, and the like. For example, when the desired protein is a basic peptide, it may be used at 1-10 mg / mol.

By column chromatography of the hydroxylamine reactant once more, a basic peptide or basic protein of at least 95% purity can be obtained in a yield of about 60%.

Example 1 Production of Merginine Derivative Fusion Proteins

In order to produce the merginine derivative MSI-344 (FIG. 1) in the form of a fusion protein, as described in Korean Patent Application No. 1999-17920, a DNA construct obtained by fusion of MSI-344 to F4, a fusion partner, was used as Nde I of pGNX2. And transformed into Bam H I locus obtained. The resulting transformants were cultured in R medium added with cassamino acid. Peptide expression was induced by adding 2 mM IPTG when the culture was between OD ₆₀₀ 0.2-0.4. To the culture medium, 0.5 mM of phosphate dehydrogenase PMSF (Phenylmethylsulfonyl fluoride) and EDTA (Ethylene Diamine Tetraacetic Acid) were added and mixed in 1L. This was passed twice at 1000 bar using a microfluidizer, a cell breaker. Microscopically, more than 99% of the cells were disrupted.

The crushed culture was centrifuged (5000 g, 5000 rpm / 30 minutes) to recover insolubles. To 200 g of the insoluble solution, add 1 L of PBS buffer (based on 1L: NaCl 8g + KCl 0.2g + Na ₂ HPO ₄ 1.44g + KH ₂ PO ₄ 0.24g), adjust the pH to 7.4, and add water to the final 1L. 1L was added and stirred until it was completely homogeneous. The pH was adjusted to 5.0 and centrifuged (5000 g, 5000 rpm / 30 minutes). To this was added 50 mM phosphate buffer to 1 L, the pH was adjusted to 5.0 and centrifuged to recover approximately 170 g of insoluble material. Per 200 mg of insoluble, 1 ml of a lysis solution containing 20 mM benzamidine hydrochloride (9M urea + 20 mM phosphate buffer (pH6.5) + 5 mM EDTA + 1% β-mulcetoethanol (pH6.5)) was added and at room temperature It was dissolved with stirring. Centrifugation removed undissolved material. 170 g of insoluble was dissolved in 1400 ml of a dissolution solution to obtain about 1550 ml of insoluble solution.

The column was filled with SP-Sepharose FF resin (Pharmacia Biotech) to obtain a bed volume of 500 ml and a bed height of 10 cm, and the elution solution (6M urea, 50 mM phosphate buffer, pH 8.5) was flowed to equilibrate the conditions in the column. 1550 ml of the insoluble solution prepared above was loaded at a linear speed of 0.6 cm / min (elution rate of 30 ml / min), and then 1000 ml of washing buffer (6 M urea, 20 mM phosphate buffer, pH8.5) was flowed at an elution rate of 50 ml / min. The impurities were removed. Again, 1000 ml of wash buffer (6 M urea, 20 mM phosphate buffer, 0.1 M NaCl, pH 8.5) was flowed at a linear speed of 1.0 cm / min (elution rate of 50 ml / min) to remove impurities. 1000 ml of the elution solution (6 M urea, 20 mM phosphate buffer, 0.3 M NaCl, pH 8.5) was flowed at a linear speed of 1.0 cm / min to obtain a fusion protein fraction. The fusion protein fraction was concentrated by ultrafiltration (MWCO = 10,000, Millipore) so that the protein concentration in the solution was about 30 mg / ml. Through this process, a fusion protein having a purity of 60-70% was obtained in a yield of 90% from the fermentation broth. Standard curves were prepared using chemically synthesized MSI-344, and the amount of MSI-344 was measured based on the prepared standard curves.

Example 2. Hydroxylamine Cleavage Reaction

The fusion protein obtained in Example 1 was prepared in a solution of 6 M urea, 0.3 M NaCl, pH 7.8-8.0 at a concentration of 20 mg / ml fusion protein. A dissolution solution for dissolving the fusion protein (43 g of guanidine HCl + 2.66 g of TRIZMA BASE + 28.3 ml of hydroxylamine HCl 6M solution and 10 N NaOH solution was adjusted to pH 8.1 and 9.5, respectively) was made for each pH. The fusion protein was dissolved in the dissolution solution such that the ratio of fusion protein to hydroxylamine was as shown in Table 1, followed by stirring in a 45 ° C. water bath for 21 hours. The reaction yield was confirmed using HPLC under the conditions described below, and the results are shown in Table 1.

TABLE 1

Reaction concentration 8.2 mg / mol 5.5 mg / mol 4.1 mg / mol PH 8.1 9.5 8.1 9.5 8.1 9.5 Yield of reaction (%) 75 63 90 80 85 75

MSI-344 protein analysis conditions after hydroxylamine cleavage reaction are as follows.

1) Column: CAPCELLPAK C18 (Shiseido)

2) Elution rate: 200µl / min

3) Injection volume: 6 μl

4) Eluent: Concentration gradient of 0.1% trifluoroacetic acid (TFA) (A) in H ₂ O and 0.1% TFA (B) in acetonitrile (0 min A: B = 85: 15, 48 min A: B = 40: 60, 55 minutes A: B = 40:60, 56 minutes A: B = 85: 15, 70 minutes A: B = 85: 15, v / v)

Example 3.

The fusion protein obtained in Example 1 was prepared in 6M urea, 0.3M NaCl, pH 7.8-8.0 solution at a fusion protein concentration of 20 mg / ml, and the fusion protein ratio to hydroxylamine was dissolved in a solution of pH 8.1 prepared above. This was dissolved to 6 mg / mol. MSI-344 yield was confirmed using HPLC while reacting in a 45 ° C. water bath over 0 to 50 hours. MSI-344 yield was good when the reaction time was 10-24 hours. The result of HPLC reaction of the reaction after 21 hours is shown in FIG. MSI-344 yield was 80%.

The antimicrobial peptide MSI-344 fraction obtained by cleaving the fusion protein with hydroxylamine was ultrafiltration (MWCO = 1000, Millipore) to remove salts. After salt removal, the antimicrobial peptide MSI-344 fractions were ion exchange chromatography. The column was filled with SP-Sepharose FF resin (Pharmacia Biotech) to have a bed volume of 400 ml and a bed height of 20 cm, and a solution of eluent (6 M urea, 30 mM potassium phosphate, pH 7.8) was flowed to equilibrate the conditions in the column. 200 ml of desalted solution was loaded at a linear speed of 0.2 cm / min (flow rate: 8 ml / min) and then 1000 ml of wash buffer (6 M urea, 30 mM potassium phosphate, 0.3 M NaCl) to remove unbound impurities present in the column. Was flowed at a flow rate of 8 ml / min. 1000 ml of the elution solution (6 M urea, 30 mM potassium phosphate, 0.5 M NaCl) was flowed at a flow rate of 8 ml / min to obtain an antimicrobial peptide fraction. The solution was lyophilized after desalting until conductance 1 ms / cm using gel filtration chromatography in 30 mM sodium acetate buffer (pH 4.5). The obtained MSI-344 purity was about 95% as measured by HPLC and the yield was 90%.

Example 4. Direct cleavage of fusion proteins using hydroxylamine

In order to produce the basic peptides, Buporin II and IIb in the form of a fusion protein, as described in Korean Patent Application No. 1999-17920, a DNA construct obtained by fusion of Buporin II and IIb to F4, a fusion partner, respectively, was used as Nde I The Bam H I site was cloned to obtain a transformant. The resulting transformants were cultured in R medium added with cassamino acid. Peptide (buforin II, IIb) expression was induced by adding 2 mM IPTG when the culture was between OD ₆₀₀ 0.2-0.4.

Cells were disrupted and insolubles were recovered in the same manner as in Example 1. About 150 g of insolubles were recovered for Buporin II and about 170 g of insolubles for Buporin IIb.

150 g of each insoluble was suspended in PBS buffer to a final volume of 750 ml. 1.5 L of each insoluble suspension was added to the reaction solution (0.22 M TRIZMA BASE + 1.7 M hydroxyamine HCl + 4.5 M guanidine HCl + methanol 110 ml, pH 8.1), followed by stirring at 45 ° C. for 24 hours, followed by HPLC. As a result, Buporin II was about 90% (FIG. 2) and Buporin IIb was about 70% (FIG. 3).

Analytical conditions of Buporin II and IIb after hydroxylamine cleavage were as follows.

1) Column: CAPCELLPAK C18 (Shiseido)

2) Elution rate: 200µl / min

3) Injection volume: 8 μl

4) Elution solvent: gradient of 0.1% trifluoroacetic acid (TFA) in H ₂ O (A) and 0.1% TFA (B) in acetonitrile (0 min A: B = 85: 15, 5 min A: B = 85: 15, 35 minutes A: B = 15: 85, 40 minutes A: B = 15: 85, 41 minutes A: B = 85: 15, 50 minutes A: B = 85: 15, v / v)

According to the present invention, a basic peptide can be obtained from a fusion protein including a basic peptide in a yield of about 95 and about 90% by a simple process.

Claims (4)

(correction) From a fusion protein, a basic peptide having a pi of 10 or more, or a fusion protein having a hydroxylamine cleavage site between a basic protein and a fusion partner is reacted with hydroxylamine at pH 7.5-8.5, and then the basic peptide is recovered from the reactant. A method of recovering a basic peptide or basic protein with a pi of 10 or more. The method of claim 1, wherein the ratio of the fusion protein and hydroxylamine is 1-10 mg of fusion protein per mol of hydroxyamine. The method of claim 1, wherein the basic peptide is merginine, buporin, defensin, histone, or derivatives thereof. (correction) Collecting the fusion protein from the fermentation broth or cells; Reacting the collected fusion protein with hydroxylamine at pH 7.5-8.5; And recovering the basic peptide or basic protein having a pI of 10 or more from the reactant, wherein the basic peptide or basic protein purifying of a pI of 10 or more.