JPH02186996A - Purification of human bcdf - Google Patents

Abstract

PURPOSE:To readily and massively provide the subject substance purified in a high purity without containing endotoxin by subjecting a solution containing a human B cell differentiation factor produced by Escherichia coli to two step reverse phase chromatography. CONSTITUTION:A solution containing a human B cell differentiation factor (human BCDF) obtained from Escherichia coli is subjected to two step reverse phase chromatography to provide the purified human BCDF having a protein purity of >=99% and an endotoxin purity of <=0.6 endotoxin unit/mg protein. The crude human BCDF as the raw material includes a fused protein which is a human BCDF having a secretion signal peptide at the N terminal thereof.

Description

DETAILED DESCRIPTION OF THE INVENTION <Industrial Application Field> The present invention relates to a method for purifying human B cell differentiation factor (hereinafter referred to as human BCDF).

<Prior art> Mature B cells activated by antigen stimulation divide and proliferate with the help of T cells, but in addition B1! ! It is known that one or more mononuclear cell-derived differentiation-inducing substances are essential for the final differentiation of cells into antibody-producing cells. The existence of this substance is RlW, Dutto
n et al. (Transplant, Rev, 2366
(1975)), A. Schimpl and W.
Echer et al. (NatureN, Biol, 23
7.15 (1972)).

Subsequently, functional evidence indicating the existence of such factors has been accumulated in mice and humans, and it is now possible to identify factors that differentiate cells into antibody-producing cells using BI.
It has come to be collectively called I cell differentiation factor CDF).

The present inventors conducted further research and determined the DNA and amino acid sequences of BCDF (Japanese Unexamined Patent Publication No. 6
3-42688, JP-A-63-56291) Successful production of human BCDF by L Escherichia coli (JP-A-63-56291)
-157996).

It has also been found that BC leaves can be used as an effective immunotherapeutic agent for the treatment of infectious diseases and cancer. (62-289007)
It has also been proposed that human BCDF be called BSF-2 or interleukin 6 (It, -6);
(Nature, 324.73 (1986),
EMBO, J., 61219 (1987)) The conventional name BCDI' is used here.

Now, this human BC leaf has a highly hydrophobic molecule, and cannot be sufficiently recovered using a purification method that combines intrashell ion exchange and gel filtration.

Furthermore, when produced using Escherichia coli, it was difficult to highly remove proteins derived from Escherichia coli and the pyrogen endotoxin (lipopolysaccharide).

Therefore, there is a long-awaited need for an efficient purification method for producing BCDF using Escherichia coli.

Problems to be Solved by the Present Invention An object of the present invention is to provide a method for easily purifying a large amount of endotoxin-free, highly purified human BCDF.

Means for Solving the Problems> In order to solve the above problems, the present inventors have incorporated the Hi1-BC leaf gene into the Tsutsunocoli bacteria, which have been extensively studied, and cultivated them in large quantities. crude human BCDF in medium or culture supernatant
The present invention was completed based on the discovery that a protein can be purified into highly purified BC protein that does not contain endotoxin by performing two steps of reversed phase chromatography.

The invention will be explained in detail below.

For example, the following can be used as the crude BCDF used as a raw material for purification.

That is, ■ Phe-Arg- at the N-terminus of the human BCDF protein.
with the N-terminal fragment (21 amino acids) of human interleukin-2 through three amino acids of 81a,
Monokugaiuchi et al., J., Biochem.

104, 30-34 (8B), JP-A-63-157
996), ■ human BC protein itself or its N-terminus with Net added, which accumulates in the bacterial body as insoluble granules or soluble fraction, ■ human BCDF protein with a secretion signal at its N-terminus. There are fusion proteins with peptides.

Each of the above raw materials is solubilized and purified using an appropriate method. For example, for (2), insoluble granules were obtained according to a conventional method, and then solubilized with a denaturing agent mainly consisting of 6M guanidine hydrochloride.
(1987)) to convert the intramolecular disulfide bond into the active form.

At that time, the protein concentration of BCDF is 50 to 300 μg.
/ml, preferably 75 to 120 μg/ff11, and purity is 5%, preferably 30% or more.

After conversion, it was concentrated directly or by ultrafiltration, and then subjected to gel filtration using Sephadex G-25 to obtain a solution for enzyme digestion reaction (50 IRM Tris-HCl, 56 mM NaC!, p
Replace with H8,1). 0.06 to 0.0% per 1 kg of fusion Hi1-BC leaf protein. 1 5 U of human plasma kallikrein (Sigma) is added and incubated at 37° C. for 15 hours (Japanese Patent Application Laid-Open No. 157996/1986). After the reaction, human IL-27 fragment (21
After confirming that the a) and Phe-Arg sequences have been cleaved, trifluoroacetic acid (TPA) is added to a final concentration of 0.3%. This solution is stable for more than one month at 4°C.

This is chemically bonded (C8) silica gel (particle size 15-2
0 μm) as a packing material and eluted with an acetonitrile concentration gradient. The obtained Hi-1-BC leaf fraction was subjected to cooling phase separation (Japanese Patent Application No. 62-23928
9), and after sufficiently diluting with o, i% TFA (acetonitrile concentration 20% or less), chemically bonded (C8)
Reverse phase with silica gel (particle size 10 μm) as filler) I
It is applied to a PLC column and similarly eluted with an acetonitrile concentration gradient. The protein purity of the obtained human BCDF was 99
% or more (polyacrylamide electrophoresis, reversed phase 1 (PLC)
, and gas phase sequencer), endotoxin was 0.6 endotoxin units (El)/■ human BCD
F or less (according to Seikagaku LAL assay kit, Toxicate System), the recovery rate is about 20 to 40%. If necessary, continue with Sephadex G25.
a suitable aqueous solution by gel filtration or lyophilization using
Alternatively, it can be made into a dry powder.

For (2), solubilize the insoluble granules in the same manner as (2), or homogenize all the cells, filter the denatured gel, and replace with 0.1% TFA solution.

The following is a two-step reversed phase similar to ①) By performing IPLC, pure human BCDF that does not contain endotoxin can be obtained.

Regarding (2), the bacterial cells collected by continuous centrifugation are disrupted by the osmotic shock method according to a conventional method to obtain highly pure crude human BCDF. Since all the N-terminal amino acid sequences and disulfide bonds are the same as in the active form, pure human BCDF free of endotoxin can be obtained by directly performing two-step reverse phase HPLC similar to ① and ②.

Hereinafter, the present invention will be specifically explained with reference to Examples.

<Example 1> Phe-Arg- at the N-terminus of the human 8C leaf amino acid sequence
DNA encoding a fusion protein in which Ala was added and a 21-amino acid sequence from the N-terminus of human interleukin 2 was added upstream of the DNA was incubated in Escherichia coli HBIOI (Bsch
-erichia coli AJ 12322,
FERM BP-1404, JP-A-63-157996
(see).

This was cultured using M9 casamino acid medium and (6ff)
,) By inducing the ributofupn promoter with indole acrylic acid (IAA), a significant amount of the above fused human BCDF protein was accumulated in the bacterial cells as insoluble granules. After solubilizing the granules according to a conventional method (Japanese Unexamined Patent Publication No. 61-257931), oxidized Gluguti 1 fluoride, 2M, and the above fused human BCDF, 300 g/ml, were added.
50mM) Liss hydrochloride, 5 6mM NaC (1
, pH8. Sephadex G-25 equilibrated to 1
The protein fraction (3.5β,
450 Akebono protein present) was obtained. To this was added 45 units of human white rice kallikrein (manufactured by Sigma), and the mixture was incubated at 37°C for 15 hours, and excess fragments were cut and removed.

While stirring the solution, add 20% trifluoroacetic acid (TFA) 5 2. Add 5 ml dropwise to a final concentration of 0. Eight rounds were made at 3%. Add this to 0.1% TFA,
Reversed phase HPLC equilibrated with 32% acetonitrile
It was loaded onto a column (YMC. R-855-15-30 Yamamura Kagaku Kenkyu Shitsu) and eluted with an acetonitrile concentration gradient (Figure 1). The obtained human BCDF fraction (0
．． 3Z) was concentrated by cooling phase separation (Tokukou Sho 62-239289), and an equal amount of 0 and 1% TFA was added to sufficiently lower the acetonitrile concentration.

This was equilibrated with 0.1% TFA and 32% acetonitrile to a reverse phase 11PLc column (YMC.AP-243).
-10 Yamamura Chemical Research Fracture) was loaded with 176 amounts each,
A total of 270 mg of BCDF fraction manufactured by n.

The chromatogram at this time is shown in FIG.

The protein purity of the thus obtained purified BCDF fraction was 99% or more (by reversed phase IPLC, 5OS-polyacrylamide electrophoresis, and gas phase sequencer),
Endotoxin is less than 0.6 EU/mg human BCDP (
(Seikagaku LAL Assay Kit, Toxicolor System), and the purification recovery rate was approximately 36%.

Effectiveness〉 When the purification method of the present invention is used, H-B produced with E. coli can be
CDF can be purified easily and efficiently.

[Brief explanation of the drawing]

FIG. 1 is the first reversed phase HPLC chromatogram. Figure 2 is the second-stage reversed phase 1 (PLC chromatogram).

Claims (1)

[Claims] 1) In purification on human BCDF produced in E. coli,
A solution containing human BCDF obtained from Escherichia coli is subjected to two-step reversed phase chromatography to purify human BCDF to a protein purity of 99% or more and endotoxin to 0.6 endotoxin units (EU)/mg protein or less. A method for purifying human BCDF.