KR0185330B1 - Method of manufacturing tumor necrosis factor in e. coli - Google Patents

Abstract

In the present invention, E. coli is transformed simultaneously with an expression plasmid of a tumor necrosis factor gene and a chaperone gene expression plasmid, followed by culturing the transformant and expressing the chaperone gene by thermal shock, thereby overexpressing the tumor necrosis factor. It relates to a method for producing a soluble tumor necrosis factor.

Description

Method for Preparation of Soluble Human Tumor Necrosis Factor in Escherichia Coli

1 is the preparation of plasmids pACTNF and pACM3S.

2 is a schematic diagram of plasmids pOF39 and pACTNF.

Figure 3 shows the solubility of overexpressed wild type tumor necrosis factor and variant (M3S) when GroEL and GroES are expressed together (a) and not (b). Where T is the whole cell eluate, S is the supernatant of the cell eluate, and P is the pellet of the cell eluate.

The present invention relates to a method for preparing a soluble human tumor necrosis factor (hereinafter abbreviated as TNF) having biological activity in E. coli.

In particular, the present invention relates to a method of preparing wild-type TNF and its variants M3S in a biologically active state by expressing wild-type human TNF and its variants M3S together with Chefrons (GroEL and GroES) in E. coli.

Tumor necrosis factor is a kind of cytokine secreted by monocytes and macrophages by stimulation of lipopolysaccharide, the cell wall constituent of Gram-negative bacteria, and is applied to various types of cancer cells in vivo and in vitro. Have cytostatic and cytotoxic effects (Playfair JH Taverne, J. in Tumor Necrosis Factor and Related Cytotoxins 192-205, 1987).

Tumor necrosis factor also has antiviral action, is known to have a prophylactic effect against malaria infection, and the scope of application is increasing.

Various methods have been attempted to obtain human TNF, and at first, the purified product was separated and purified in human body, but a sufficient amount could not be obtained. Recently, however, mass production has become possible thanks to the development of genetic engineering methods.

The gene of human tumor necrosis factor was cloned and expressed in E. coli in 1984, and has a long anti-parallel β-sheet sandwich structure with a jelly-roll topology, 3 It exists as a trimer with 3-fold symmetry and has one disulfide bond (Aggarwal, BB et al., J. Biol. Chem. 260, 2345-2354, 1985).

Tumor necrosis factor is difficult to apply to the actual cancer treatment because it can not administer the appropriate amount that can have an anticancer effect due to side effects. Protein mutations have been attempted to create tumor necrosis factor muteins with increased anti-cancer effects and reduced side effects than wild-type tumor necrosis factor.

The present inventors produced mutein M3 by substituting isoleucine and phenylalanine for serine and tyrosine at amino acid sequence 52 and 56th of tumor necrosis factor among these muteins, respectively. In the case of mutein M3, the anticancer effect was increased by 30 times compared to the natural type.

However, the mutein M3 is four times more adverse than wild type. Accordingly, the present inventors have filed a patent application for obtaining the M3S which significantly reduced the toxicity while maintaining the high anticancer effect of M3 by replacing the 29th leucine in mutein M3 with serine (Patent Application No. 95-2752). .

E. coli is mainly used as a host in the production of human tumor necrosis factor. E. coli is most frequently used for mass production of TNF due to its short passage time and simple culture conditions.

However, when the expression of human TNF in E. coli is produced as a solid body (inclusion body), the yield is reduced by the continuous process, such as re-folding to obtain a biologically active TNF from the TNF obtained as a solid body, the yield is reduced by the continuous treatment .

In some cases, the growth conditions such as temperature and the composition of the medium can be avoided to some extent, but the general conditions for preventing the formation of solids are not known. Therefore, the present inventors have studied the method of preparing human TNF and its variant M3S in the dissolved state in E. coli, and found that when expressed together with chaperon protein, soluble human TNF and its variant M3S are produced. Completed.

It is an object of the present invention to provide a method for preparing soluble TNF and its variant M3S with biological activity by expressing TNF in a dissolved state when human TNF and its variant M3S are expressed in E. coli.

Hereinafter, the present invention will be described in detail.

Genetic information of living organisms is the transmission of genetic information from linear genomic DNA to proteins of three-dimensional structure. Until recently, only RNA transcription, RNA maturation, and protein translation were considered important in this communication, but recent studies have shown that post-translational modifications are important for polypeptides to have biological activity. That is, polypeptides produced by deciphering RNA have different life spans and modification processes according to their respective characteristics, and expression rates may be controlled by intracellular conditions.

In various modifications, the folding of the polypeptide requires an accessory factor, and in particular, a particular protein called chaperone has been found to play an important role in folding during post-translational modification. Without the chaperone protein, the correct folding does not occur, and if the correct folding does not occur, aggregation of the protein molecules occurs, solubility is reduced and becomes a solid. Schaeffron is a heat shock protein, a protein whose expression is increased by heat shock, and functions to make a polypeptide that has changed to an abnormal structure at a high temperature again into a normal structure.

The protein can be produced in Escherichia coli and has been reported to increase the solubility of the recombinant protein in vivo and ex vivo.

There are two types of chaperon proteins, thermal shock protein 60 called GroEL and thermal shock protein 10 called GroES. These proteins are expressed at room temperature and are overexpressed due to increased expression when subjected to thermal shock.

GroEL is a high molecular weight complex that hydrolyzes ATP as a K ⁺ dependent ATPase. It is composed of 14 subunits corresponding to 60KDa and is stacked in two rows of seven. In particular, GroEL is called chaperonin, and it functions by combining with GroES.

Cheppronin is a ribosome-sized ring-shaped oligomer that binds sequentially to abnormal polypeptides and is released by ATP hydrolysis to allow for proper folding into normal polypeptides.

There are two models in which GroE proteins help correct protein folding. The first model involves the chaperone binding to the newly formed polypeptide chain and directly involved in the morphology of the protein. The second model does not directly promote the correct folding of the chaperone, but only binds the polypeptide to prevent aggregation of abnormal polypeptides. To prevent.

In addition to the important role Schafflong plays in the folding of proteins, it has recently been reported that overexpression with GroEL and other proteins intended to express GroES increases the activity of the desired protein by GroEL and GroES proteins. .

Based on the above report, the present inventors focused their efforts on obtaining increased activity of TNF and variants (M3S). When TNF coexpresses human tumor necrosis factor with Chef (GroEL and GroES), most of the TNF is not obtained as a solid. The present invention was completed by finding that it was obtained in a dissolved state (Glenn E. Dale, et al., Protein Engineering vol. 7 No. 7 pp. 925-931, 1994; Arthur L. Horwich, et al., Cell 74, 909-917, Sept. 10, 1993; Johannes Buchner, et al., Biochemistry 1991, 30. 1586-1591; Alexander Gragerov, et al., Proc. Natl. Acad. Sci., USA vol. 4 pp. 10341-10344, Nov. 1992).

The present invention relates to a method for preparing soluble TNF by expressing the chaperones (GroEL and GroES) with the gene of wild type TNF or variant M3S thereof.

Hereinafter, the present invention will be described in more detail.

Wild-type human TNF or variant M3S gene is cloned into plasmid vector pACYC184 to prepare pACTNF or pACM3S. Plasmid pACYC184 has a gene region that is resistant to chloramphenicol and has a replication origin of p15A and can coexist with vectors such as pBR322 and pUC19 that have a ColE1 replication origin. The plasmid pACYC184 was digested with restriction enzymes, and then plasmids containing the TNF gene and M3S gene were inserted therein to obtain pACTNF and aggregated pACM3S, respectively.

The plasmid pOF39, which has genes that make it resistant to protein aggregation by helping to fold proteins and prevents protein aggregation and resistance to ampicillin, was obtained from the University of Utah Medical Center [Mol. Gen. (1986) 202: 435-445.

E. coli BL21 (DE3) strains were transformed with pACTNF and pOF39 or pACM3S and pOF39 by Hanahan method to obtain respective transformants.

After culturing BL21 (DE3) containing two plasmids in the medium containing ampicillin and chloramphenicol from 37 ° C. to the early log phase, the temperature is raised to 42 ° C., and then the chaperon protein is expressed. .

Next, it is induced by IPTG (isopropyl-β-D-thiogalactotypanoside, final concentration of 0.5 mM) to express a large amount of tumor necrosis factor.

Thus, when the chaperone is first expressed and then the tumor necrosis factor is expressed, both wild-type and variants are obtained as a protein in a dissolved state of 95% or more.

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

Example 1

Preparation of pT7-TNF

Human tumor necrosis factor-alphaDNA was synthesized from the cDNA library by PCR and double-cut with NdeI and BamHI enzymes, respectively. The expression vector system for this was used pT7-7 (available from the Department of Biochemistry, Harvard Medical School) having a T7 expression system and removing the T7 gene fusion region. pT7-7 was double-cleaved with NdeI and BamHI enzymes, and the expression vector plasmid pT7-TNF was obtained by linking the vector and tumor necrosis factor DNA cleaved by the two enzymes described above with T ₄ DNA ligase.

Example 2

Preparation of pT7-M3S

PT7-M3S was prepared in the same manner as in Example 1. M3S is a variant wherein seven amino acids at the amino terminus are removed from the amino acid sequence of TNF and the 29th leucine is substituted with serine, the 52nd serine is substituted with isoleucine, and the 56th tyrosine is substituted with phenylalanine.

Example 3

Preparation of pACTNF and pACM3S

The plasmid pACYC184 was digested with restriction enzymes BamHI and HindIII, and then a vector portion of 3.9 kb was isolated. In addition to separate a DNA fragment of about 600bp containing the wild-type tumor necrosis factor (TNF) and its variant (M3S) gene and the T ₇ expression system by cutting the plasmid pT7-TNF and pT7-M3S with restriction enzyme BalII and HindIII.

To the 3.9 kb vector prepared from plasmid pACYC184, 600 bp DNA fragments isolated from plasmid pT7-TNF or pT7-M3S were linked using T ₄ DNA ligase enzyme, respectively.

Each plasmid made was named pACTNF and pACM3S (see Figure 1).

Example 4

Preparation of Transformant

Transformants were obtained by transforming E. coli BL21 (DE3) strains simultaneously with pACTNF, a wild-type human TNF expression vector, and pOF39, a chaperone expression vector.

Variant E.heri coli BL21 (DE3 + pOF39 + pACM3S) was transformed by simultaneously transforming E. coli BL21 (DE3) strain with the M3S expression vector pACM3S and the chaperone expression vector pOF39. Recorded in association (Accession No. KCCM-10075).

Example 5

Culture of transformants

Colonies resistant to ampicillin and chloramphenicol were picked out, inoculated into LB culture medium containing chloramphenicol and ampicillin, incubated at 37 ° C. to an early log phase, and then incubated at 42 ° C. for 30 minutes. GroEL and GroES were first expressed in thermal shock.

IPTG was added at an optimal concentration of 0.5 mM and shaken at 37 ° C. for 3 hours to express TNF in excess.

Example 6

Experimental Studies on Solubility of Wild-type TNF and Variants M3S Overexpressed When GroEL 롸 GroES Coexpressed (a) and Not (b)

The culture solution was centrifuged to obtain E. coli pellets, suspended in 10 mM sodium phosphate buffer (pH 7.6), and the cells were pulverized by sonication.

The pulverized cell solution was again centrifuged and separated into a supernatant and a pellet, and the solubility of TNF and M3S that was overexpressed when GroEL and GroES were not expressed together using SDS-PAGE was compared. 3).

When E. coli expresses wild-type human tumor necrosis factor (TNF) or its variants (M3S) using T expression system, about 30% of wild-type human TNF and about 60% of variant M3S are obtained as a solid. To obtain wild type human TNF or variant M3S with biological activity, TNF obtained as a solid must be dissolved or reconstituted.

However, as can be seen from the above experimental results, it can be seen that according to the method of the present invention, the resulting TNF or a variant thereof is prepared in a dissolved state of 95% or more.

Therefore, since the TNF or a variant thereof produced by the preparation method of the present invention is obtained in a dissolved state, there is no need for reconstitution, and since it is obtained in a state where biological activity is correctly folded from the beginning, the yield is increased.

[Sequence list]

SEQ ID NO: 1

Length of sequence: 157

Type of sequence: amino acid

Topology: linear

Type of molecule: peptide

[SEQ ID NO 1]

Claims (8)

A method for producing soluble tumor necrosis factor obtained by dissolving tumor necrosis factor in a dissolved state by simultaneously transforming host cells with expression plasmids of tumor necrosis factor gene and expression plasmids (Chaperones; GroEL and GroES). The method of claim 1, wherein the tumor necrosis factor is a wild type human tumor necrosis factor. The method of claim 1, wherein the tumor necrosis factor is variant M3S. Expression plasmid pACTNF having a cleavage map of FIG. 2 with a plasmid containing a gene of wild type human tumor necrosis factor. An expression plasmid pACM3S containing a gene of variant M3S of wild type human tumor necrosis factor, wherein the gene of variant M3S was inserted in place of wild type TNF in the cleavage map of pACTNF of FIG. A transformant transformed with E. coli BL21 (DE3) simultaneously with the expression plasmid pACTNF of the tumor necrosis factor gene and the expression plasmid pOF39 of the chaperone gene. A transformant transformed with E. coli BL21 (DE3) simultaneously with the expression plasmid pACM3S of the tumor necrosis factor gene and the expression plasmid pOF39 of the chaperone gene (Accession No. KCCM-10075). The method of producing a soluble tumor necrosis factor, characterized in that the culture of the transformant of claim 6 or 7, and then subjected to thermal shock first to express the chaperone, and then induced by IPTG in excess of the tumor necrosis factor.