WO2000034312A1

WO2000034312A1 - Method of separating basic peptide or basic protein

Info

Publication number: WO2000034312A1
Application number: PCT/KR1999/000748
Authority: WO
Inventors: Heung-Bok Park; Sang-Hyun Pyo; Sung Wook Hwang; Jin-Eon So; Jin-Hyun Kim; Jeong Hyun Kim; Seung-Suh Hong; Hyun-Soo Lee
Original assignee: Samyang Genex Corporation
Priority date: 1998-12-10
Filing date: 1999-12-08
Publication date: 2000-06-15
Also published as: AU1805200A

Abstract

The present invention relates to a method of recovering basic peptide or basic protein at a high yield from a fusion protein that has a hydroxylamine cleavage site between the basic peptide or basic protein and the fusion partner. More particularly, the present invention is composed of the processes of reacting fusion protein with hydroxylamine at a pH of 7.5 ∩ 8.5 and recovering the basic peptide from the reaction mixture.

Description

METHOD OF SEPARATING BASIC PEPTIDE OR BASIC PROTEIN

TECHNICAL FIELD

The present invention relates to a purification and recovery method for a

basic peptide or a basic protein from recombinant cells.

BACKGROUND ART

Many peptides or proteins that have biological activities such as

antimicrobial or cytotoxic activity are basic peptides. Generally, it is very difficult

to express basic peptides or basic proteins in recombinant cells at a high

expression rate. The expression rate is low because the produced peptides

degrads rapidly in the cells, or the host cells die due to the cytotoxicity of the

produced peptides. Also it is difficult to separate the peptides from other proteins

present in the cell. To resolve these problems, peptides to be separated are

expressed as fusion proteins.

Expressing a peptide in the form of a fusion peptide have advantages of

preventing degradation of the produced peptides in the cell or of avoiding death of

the host cells due to the cytotoxicity of the produced peptide. However, the yield

of the proteins or peptide after separation from the fusion protein is extremely low.

One method to increase the yield of the peptide is to introduce a proper recognition sequence between the fusion partner and peptide in the fusion protein

or fusion peptide By cleaving the peptide from the fusion partner the peptide

from the fusion protein can be separated This cleavage method includes an

enzymatic method using enzymes and a chemical method using chemical

substances

Chemicals used in the chemical cleavage method are CNBr and

hydroxylamine The yield of the peptides or proteins, however is as low as ca

40 % by using the chemical method Antonni et al (Protein Expression and

Purification 11 , 135-147, 1997) reported the conditions that influence the yield of

the peptide when a fusion protein having hydroxylamine cleavage site is purified

by digestion with hydroxylamine It was reported that the reaction pH is one of the

important reaction conditions and that the protein recovery rate is high at a high

pH The optimal pH of the reaction was suggested to be 9 5 They obtained

IGF-I at ca 50-60 % yield by cleaving the fusion peptide at a low concentration of

fusion peptide, 1 -4 mg/M at pH 9 5

DISCLOSURE OF THE INVENTION

The present invention provides a method of separating basic peptide

effectively from a fusion protein containing a hydroxylamine cleavage site and

basic peptide

The present invention also provides a method of purifying basic peptide at a high yield from recombinant cells.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a chromatogram showing the cleavage of fused MSI-344 by a

hydroxylamine reaction.

Figure 2 is a chromatogram showing the cleavage of fused buforin II by a

hydroxylamine reaction.

Figure 3 is a chromatogram showing the cleavage of fused buforin lib by a

hydroxylamine reaction.

DETAILED DESCRIPTION OF THE INVENTION

A fusion protein in the present invention represents the fusion protein that

has a hydroxylamine cleavage site between a fusion partner and a foreign basic

peptide or basic protein.

The present inventors have found that the peptide or protein recovery rate

can be highly enhanced by adjusting the reaction pH to 7.5~ 8.5 which is lower

than the reported pH of 9.5 which was suggested by the conventional technique in

the case that the foreign peptide or protein to be recovered is basic.

The present invention relates to a method of recovering basic peptide or

basic protein from a fusion protein. The recovery method according to the present invention comprises the

steps of reacting a fusion protein having a hydroxylamine cleavage site between a

fusion partner and a foreign basic peptide or basic protein with hydroxylamine at a

pH of 7.5~8.5 and recovering the basic peptide from the reaction products.

The present invention relates to a method of purifying peptide at a high

yield from recombinant cells.

The purification method according to the present invention comprises the

following steps: recovering cells from a fermentation medium; collecting fusion

proteins which comprises fusion partner and basic peptide or protein from cell

extracts or culture medium; reacting fusion peptides with hydroxylamine at pH

7.5~8.5; and recovering basic peptide or basic protein from the reaction mixture.

In the case the fusion protein is secreted from the cells according to the

method of the present invention, the fusion protein is collected from the reaction

medium and treated with hydroxylamine.

In the case the fusion protein remains in the cells according to the method

of the present invention, the fusion protein is collected from the cell extracts and

treated with hydroxylamine.

The reaction time according to the present invention is 10~24 hours,

preferably 15~22 hours.

The basic peptide or basic protein can be recovered at a yield of ca. 70 ~

90 % by using the above methods of the present invention.

The method of the present invention can be used to separate basic peptide or basic protein from the fusion peptide. Examples of basic peptides

include magainin, buforin, defensin, and examples of basic proteins include

histone.

In the purification method according to the present invention, to prevent

the degradation of the peptides or proteins by proteolytic enzymes, it is preferable

to add proteolytic enzyme inhibitors. The proteolytic enzyme inhibitors that can

be used during the purification process include PMSF (Phenylmethylsulfonyl

fluoride), EDTA (Ethylene Diamine Tetraacetic Acid), benzamidine and ZnCI₂.

The amount of inhibitor can vary depending on the fusion protein, the recombinant

microorganism, or the inhibitors used.

The fusion peptide or fusion protein can be accumulated in the intraceilular

space or secreted to the culture medium depending on the peptide or the protein,

fusion partner or cells. In the case the peptide or protein is accumulated in the

intraceilular space, the separated ceils or the culture medium itself need to be

homogenized by using a sonicator, microfluidizer or mill.

The method of recovering fusion protein from the cell extract or culture

medium includes, for instance, centrifuging cell extract or culture medium or

passing through a membrane.

Sometimes, it is necessary to pre-treat the cell extract or culture medium

to remove impurities before the hydroxylation reaction of the fusion protein. For

example, a cation exchange chromatography can be used. Especially when the

fusion protein is insoluble, the fusion protein can be dissolved in urea or guanidine, and impurities can be precipitated to obtain the fusion protein at an increased

purity.

When the insoluble protein is recovered by passing through a membrane,

the composition of the membrane, the cut off value and the flow rate can be

controlled to increase the recovery rate of the insoluble protein and to eliminate

the impurities. Therefore, the method of using a membrane is preferable since

the pre-treatment process before the hydroxylation reaction can be eliminated.

The amount of hydroxylamine can vary depending on the protein, the

fusion partner and the size of fusion protein. For instance, 1~10 mg/mol can be

used when the protein to be obtained is a basic protein.

By performing an additive column chromatography for the hydroxylamine

reaction mixture, the basic peptide or basic protein can be obtained at a ca. 60 %

yield with higher than 95 % purity.

BEST MODE TO ACHIEVE THE EXAMPLES OF THE INVENTIONS

The present invention will be further illustrated by the following examples.

It will be apparent to those having conventional knowledge in the field that these

examples are given only to explain the present invention more clearly, but the

present invention is not limited to the examples given.

EXAMPLE 1. Production of Magainin Derivative Fusion Peptide To produce magainin derivative MSI-344 in the form of fusion peptide, a

transformant was obtained by cloning DNA obtained by fusing MSI-344 with F4 at

an Nde I and SamH I sites of pGNX2. The obtained transformant was cultured in

a R medium supplemented with casamino acid. Peptide expression was induced

by adding 2 mM IPTG when the OD₆₀₀ was between 0.2 and 0.4. Proteolytic

enzyme inhibitors, PMSF (Phenylmethylsulfonyl fluoride) and EDTA (Ethylene

Diamine Tetraacetic Acid) at 0.5 mM each were added in a 1 L culture medium

and mixed completely. This mixture was passed twice through a microfluidizer to

destroy cells at 1000 bars. It was confirmed by observing under a microscope

that more than 99 % of cells were homogenized.

A homogenized culture medium was centrifuged at 5000 g and 5000 rpm

for 30 minutes to recover the insoluble matter. To the 200 g of recovered

insoluble matter, 1 L PBS buffer solution (To make 1L PBS: Mix NaCI 8g + KCI 0.2

g + Na₂HPO₄ 1.44 g + KH₂PO₄ 0.24 g and water, and adjust the pH to 7.4 and add

the rest of water to make the final volume to make 1 L) was added. After the pH

was adjusted to 7.4, the solution was stirred until mixed homogeneously. The pH

was adjusted to 5.0 before centrifuging the solution at 5000 g and at 5000 rpm for

30 minutes. After this procedure, 50 mM phosphate buffer solution was added to

make total volume to 1 L and the pH was adjusted to 5.0, and the solution was

centrifuged to recover ca. 170 g of insoluble material. For 200 mg of insoluble

material, after adding 1 ml of dissolution buffer (9M urea + 20 mM phosphate

buffer solution (pH 6.5) + 5 mM EDTA + 1 % β-mercaptoethanol (pH 6.5)) containing 20 mM benzamidine hydrochloride, the solution was stirred at room

temperature for solubilization. Insoluble materials were eliminated by

centrifugation. By soiubilizing 170 g of insoluble matter in 1400 ml of dissolution

buffer, ca. 1550 ml of solution containing insoluble matter was obtained.

A column was packed with SP-Sepharose FF resin to make a bed volume

of 500 ml and a bed height of 10 cm. By flowing elution buffer (6 M urea, 50 mM

phosphate buffer solution, pH 8.5), the equilibrium was achieved inside the

column. After loading 1550 ml of the solution containing insoluble matter to

make a linear rate to 0.6 cm/min (elution rate 30 ml/min), 1000 ml of washing

buffer (6 M urea, 20 mM phosphate buffer solution, pH 8.5) was eluted at a 50

ml/min rate to eliminate impurities that did not bind on the resin. The impurities

were eliminated again by eluting 1000 ml of washing buffer (6 M urea, 20 mM

phosphate buffer solution, pH 8.5) at a linear rate of 1.0 cm/min (elution rate 50

ml/min). By flowing dissolution buffer (6 M urea, 20 mM phosphate buffer

solution, 0.3 M NaCI, pH 8.5) at a linear rate of 1.0 cm/min, the fusion protein

fraction was obtained. The fusion protein fraction was ultracentrifuged (MWCO =

10,000, Millipore) to condense the protein to a concentration of ca. 30 mg/ml. By

using the above process, the fusion protein was obtained at a 90 % yield with ca.

60-70 % purity.

EXAMPLE 2. Hydroxylamine Cleavage Reaction

The fusion protein obtained from Example 1 was mixed in 6 M urea, 0.3 M NaCI, pH 7.8 ~ 8.0 at 20 mg/ml concentration of fusion protein. To dissolve the

fusion protein, dissolution solutions (28.3 ml of guanidine HCI 43 g + TRIZMA

BASE 2.66 g + 6M hydroxylamine HCI solution; pH of the solution was adjusted to

8.1 or 9.5 by using 10 N NaOH) were prepared at different pH's. The fusion

protein was solubilized in a dissolution solution at different ratios between

hydroxylamine and the fusion protein as shown in Table 1. The solution was

stirred in a 45 °C water-bath for 21 hours. By using the condition described

below, the reaction yield was determined by using HPLC, and the results are

shown in Table 1.

Table 1

After the hydroxylamine cleavage reaction, the analysis condition of MSI-344

protein was as follows.

1) Column: CAPCELLPAK C18 (Shiseido)

2) Elution rate: 200 μl/min

3) Injection volume: 6 μl

4) Elution solvent: Concentration gradient (0 min A:B = 85:15, 48 min

A:B = 40:60, 55 min A:B = 40:60, 56 min A:B = 85:15, 70 min A:B = 85:15, v/v) between solution A and B; solution A) 0.1 % trifiuoroacetic

acid (TFA) in water and solution B) 0.1 % TFA in acetonitrile

EXAMPLE 3.

The fusion protein obtained from Example 1 was mixed in 6 M urea, 0.3 M

NaCI, pH 7.8 ~ 8.0 at a concentration of 20 mg/ml fusion protein. In a pH 8.1

dissolution solution prepared as above, the fusion protein was solubilized by

adding 6 mg of the fusion protein per 1 mole of hydroxylamine. MSI-344 was

obtained after reaction for 0~50 hours in a 45 °C water-bath. The yield of the

MSI-344 was determined. When the reaction time was 10-24 hours, the yield of

the MSI-344 was the highest. The product mixture after 21 hours reaction was

analyzed by HPLC and is shown in Figure 1. The yield of the MSI-344 was 80 %.

The MSI-344 fraction that was obtained by cleaving the fusion protein with

hydroxylamine was ultrafiltrated (MWCO = 1000, Millipore) to remove salt. After

removing the salt, antimicrobial peptide MSI-344 fraction went through ion

exchange chromatography. A column was packed with SP-Sepharose FF resin

(Pharmacia Biotech) to make a bed volume of 400 ml and a bed height of 20 cm.

By flowing elution buffer (6 M urea, 30 mM phosphate buffer solution, pH 7.8),

equilibrium was achieved inside the column. After loading 1550 ml of the

desalted solution to make a linear rate to 0.2 cm/min (elution rate 8 ml/min), 1000

ml of washing buffer (6 M urea, 30 mM potassium phosphate, 0.3 M NaCI) was

eluted at a 8 ml/min rate to eliminate impurities that did not bind on the resin. By

flowing dissolution buffer (6 M urea, 30 mM potassium phosphate, 0.5 M NaCI) at a flow rate of 8 ml/min, the antimicrobial peptide fraction was obtained. The

solution was desalted until the conductivity was below 1 ms/cm by using a gel

filtration chromatography with 30 mM sodium acetate buffer solution (pH 4.5).

The purity of the MSI-344 was ca. 95 %, and the yield was 90 %.

EXAMPLE 4. Direct Cleavage Reaction of Fusion Protein Using Hydroxylamine

To produce basic peptides, buforin II and Mb in the form of fusion peptides,

transformant was obtained by cloning DNA obtained by fusing buforin II or lib with

F4 at the Nde I and SamH I sites of pGNX2. The obtained transformant was

cultured in R medium supplemented with casamino acid. Peptide (buforin II or

lib ) expression was induced by adding 2 mM IPTG when the OD₆₀₀ was between

0.2 and 0.4. Insoluble matter was recovered by destroying cells using the

method identical as in Example 1. In the case of buforin II and lib, ca. 150 g and

170 g, respectively, of insoluble matters were recovered.

To 150 g of each recovered insoluble matter, PBS buffer solution was

added to make a total volume of 750 ml. Each suspension containing insoluble

matter (1.5 L) was added to a dissolution solution (0.22 M TRIZMA BASE + 1.7 M

hydroxylamine HCI + 4.5 M guanidine HCI + methanol 110 ml, pH 8.1). The

solution was stirred for 24 hours at 45 °C. The yield of the reaction was

determined by HPLC and was ca. 90 % for buforin II (Figure 2) and ca. 70 % for

buforin lib (Figure 3).

After the hydroxylamine cleavage reaction, the analysis condition of buforin II or lib was as follows.

1) Column: CAPCELLPAK C18 (Shiseido)

2) Elution rate: 200 μl/min

3) Injection volume: 8 μl

4) Elution solvent: Concentration gradient (0 min A:B = 85:15, 5 min A:B =

85:15, 35 min A:B = 15:85, 40 min A:B = 15:85, 41 min A:B = 85:15, 50

min A:B = 85:15, v/v) between solution A and B; solution A) 0.1 %

trifluoroacetic acid (TFA) in water and solution B) 0.1 % TFA in

acetonitrile

INDUSTRIAL APPLICATION

According to the present invention, a basic peptide can be obtained at a

purity of ca. 95 % and a yield of ca. 90 % from a fusion protein containing a basic

peptide by using a simple process.

Claims

CLAIMS;

1. A method of recovering basic peptide or basic protein from a fusion protein by

reacting the fusion protein, that has a hydroxylamine cleavage site between

the basic peptide or basic protein and fusion partner, with hydroxylamine at

pH 7.5-8.5; and recovering a basic peptide from a reaction mixture.

2. A method according to Claim 1 , wherein the ratio between the fusion protein

and hydroxylamine is in a range of 1-10 mg of the fusion protein per mole of

the hydroxylamine.

3. A method according to Claim 1 , wherein the above basic peptide is selected

from the group consisting of magainin, buforin, defensin, histone and their

derivatives.

4. A method of purifying basic peptide or basic protein comprising the following

steps:

- collecting fusion protein comprising fusion partner and the basic

peptide or basic protein from a culture medium or cells;

- reacting the collected fusion protein with hydroxylamine at pH 7.5 - 8.5;

and

recovering the basic peptide or basic protein from the reaction mixture.