阅读说明：本技术 可溶性人肿瘤坏死因子凋亡相关诱导配体的突变体及其制备方法和应用 (mutant of soluble human tumor necrosis factor apoptosis-related inducing ligand and preparation method and application thereof ) 是由 林洁 王庆华 沈飞 马鹏飞 姚丽沙 祁文瑾 于 2018-06-01 设计创作，主要内容包括：本发明属于生物技术制药领域,具体涉及可溶性人肿瘤坏死因子相关凋亡诱导配体(sTRAIL)突变体及其制备与药学应用。本发明应用定点突变技术设计并构建了系列sTRAIL突变体,并从中筛选出可溶性表达较野生型优越的新颖突变体,含有R121A、R121S、R121C或V122A中的一种或几种突变。该sTRAIL突变体较野生型sTRAIL在大肠杆菌中的可溶性表达水平有极显著提高,且其抗肿瘤活性也有一定增强。本发明还揭示了该突变蛋白的诱导表达、可溶性提取、分离纯化和加工精制等制备方法,有效克服了现有sTRIAL生产制备和药学应用中存在的问题。(The invention belongs to the field of biotechnology pharmacy, and particularly relates to a soluble human tumor necrosis factor-related apoptosis inducing ligand (sTRAIL) mutant, and preparation and pharmaceutical application thereof. The invention designs and constructs a series of sTRAIL mutants by applying site-directed mutagenesis technology, and screens out novel mutants with solubility expression superior to that of wild type, wherein the novel mutants contain one or more of R121A, R121S, R121C or V122A. Compared with wild sTRAIL, the soluble expression level of the sTRAIL mutant in escherichia coli is remarkably improved, and the anti-tumor activity of the sTRAIL mutant is also enhanced to a certain extent. The invention also discloses preparation methods of the mutant protein, such as induced expression, soluble extraction, separation and purification, processing and refining, and the like, and effectively solves the problems in the existing sTRIAL production preparation and pharmaceutical application.)

1. The mutant of human TRAIL is characterized by containing one or more mutations of R121A, R121S, R121C or V122A.

2. The mutant according to claim 1, wherein the soluble human TRAIL-related apoptosis-inducing ligand is wild-type, derived or recombinant TRAIL soluble extracellular region active fragment or membrane-bound TRAIL containing the same.

3. The mutant according to claim 2, wherein the amino acid sequence of the mutant is as shown in SEQ ID Nos. 1-4.

4. Gene coding for the mutant according to claim 3, characterized in that the mutant gene has the sequence shown in SEQ ID Nos. 5-8.

5. The method for preparing the mutant of soluble human TRAIL-related apoptosis-inducing ligand according to any one of claims 1 to 3, wherein a fusion gene comprising a gene encoding the mutant according to any one of claims 1 to 3 is prepared and expressed in a corresponding host cell.

6. The method of claim 5, comprising the steps of:

(1) Obtaining a fusion gene comprising a gene encoding the mutant of any one of claims 1-3, said fusion gene comprising three gene segments: the gene fragment at the N-end is a coding gene of a hexahistidine tag, the gene fragment at the middle part is a coding gene of a protease cutting site of the tobacco etch virus, the gene fragment at the C-end is a coding gene of a mutant according to any one of claims 1 to 3, and restriction enzyme specific recognition sites are positioned at the two ends of the gene;

(2) Cutting the fusion gene obtained in the step (1) and the vector plasmid by using the same restriction enzyme, and then connecting to obtain a recombinant expression plasmid;

(3) Transforming the recombinant expression plasmid obtained in step (2) into a host cell to form a recombinant cell capable of expressing the mutant protein of any one of claims 1-3;

(4) Culturing the recombinant cell obtained in the step (3);

(5) Extracting soluble protein from the disrupted cell fraction, and isolating and purifying to obtain a fusion protein comprising the mutant according to any one of claims 1 to 3;

(6) Processing the fusion protein obtained in the step (5) by adopting tobacco etch virus protease;

(7) Separating and purifying to obtain the mutant as claimed in any one of claims 1-3.

7. Use of a mutant according to any one of claims 1 to 3 in the manufacture of a medicament for the treatment or prevention of an apoptosis-related disease.

8. Use according to claim 7, characterized in that the medicament is an antineoplastic medicament.

9. Use according to claim 7, characterized in that the medicament comprises a mutant according to any of claims 1 to 3 as a pharmaceutically active ingredient together with a pharmaceutically acceptable carrier.

10. Use according to claim 8, characterized in that the medicament also contains a further antineoplastic drug and/or a sensitizer for the antineoplastic drug.

11. A method for soluble expression of TRAIL and its mutant features that the 121 th amino acid of TRAIL or its mutant is mutated to alanine, serine or cysteine, and/or the 122 th valine is mutated to alanine.

Technical Field

The invention belongs to the field of biotechnology pharmacy, and particularly relates to a mutant of soluble human tumor necrosis factor-related apoptosis inducing ligand (sTRAIL), which has higher soluble expression capability in a prokaryotic expression system compared with wild type TRAIL, is beneficial to improving the preparation efficiency and reducing the production cost, and simultaneously shows better anti-tumor activity than the wild type TRAIL.

Background

Apoptosis is a highly conserved programmed cell death mode in the evolution process and is an important physiological mechanism for maintaining normal development of organisms. Escape from apoptosis is a key factor for the generation, development and drug resistance of malignant tumors, and the induction of apoptosis of tumor cells becomes an important strategy for treating tumors. Death Receptor (DRs) pathways and mitochondrial pathways are currently the most effective means of inducing apoptosis. Among them, the death receptor pathway induced by Tumor Necrosis Factor (TNF) superfamily members is considered as the most promising direction for developing biological Tumor therapeutic drugs.

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) is a newly discovered TNF superfamily member, also known as Apo-2L, TNFSF10 or CD253, etc. The TRAIL-encoding gene was cloned from a cardiac cDNA library by Wiley in 1995, and the expression product was a type II transmembrane glycoprotein consisting of 281 amino acid residues. The ligand induces apoptosis of target cells by binding to its specific receptor (TRAIL receptors, TRAIL-Rs). To date, a total of 5 TRAIL-Rs of two classes have been discovered. One class is functional receptors, including TRAIL-R1 and TRAIL-R2, also called Death receptor 4 (DR 4) and Death receptor 5 (DR 5), respectively; another class is antagonistic receptors, including: TRAIL-R3 is also called Decoy receptor 1(Decoyreceptor 1, DcR1), TRAIL-R4 is also called Decoy receptor 2(Decoy receptor 2, DcR2) and soluble receptor OPG (osteoprotegerin). Among them, the functional receptors DR4 and DR5 are highly expressed in transformed cells and tumor cells, while the inhibitory receptors DcR1 and DcR2 are mainly distributed on the surface of normal cells. The diversity of TRAIL-Rs and the expression and distribution characteristics thereof determine that TRAIL has selective killing effect on various tumor cells, so that the TRAIL shows safer and more effective antitumor activity than the members of the TNF and FasL equivalent families.

The extracellular domain at the C-terminus is the active site of TRAIL, which is proteolytically cleaved into body fluids to form a soluble monomer, known as soluble TRAIL (sTRAIL). sTRAIL is able to self-assemble in vivo and in vitro to form dimers, trimers, wherein the trimeric form of sTRAIL has a biological activity comparable to that of intact TRAIL. In recent years, numerous preclinical and clinical studies show that sTRAIL can effectively induce apoptosis of various tumor cells, has no toxicity to normal tissue cells, shows therapeutic potential in diseases such as melanoma, medulloblastoma, breast tumor and glioma, and becomes an important candidate for developing a new generation of anti-tumor biotechnology drugs. TRAIL (aa114-281), TRAIL (aa 95-281) and TRAIL (aa 120-281) are the most common constitutive forms of sTRAIL.

Research shows that the sTRAIL without glycosylation modification still can show safe and effective antitumor activity, so that a prokaryotic expression system with simple operation, high yield and low cost is an optimal carrier for producing the protein and derivative products thereof. However, sTRAIL, which is an exogenous molecule, is often expressed in escherichia coli as biologically inactive inclusion bodies, and renaturation is required to obtain the final product. To date, the in vitro renaturation of proteins is still a time-consuming, costly, inefficient and difficult to scale-up procedure, which greatly limits the research, development, production and application of related products. How to effectively improve the soluble expression efficiency, optimize the production and preparation process and ensure the biological activity of the product becomes the key technology for developing a new generation of anti-tumor biotechnology medicine based on sTRAIL. The current technical approaches commonly used to increase the soluble expression efficiency of sTRAIL in E.coli are mainly based on downstream expression condition optimization with limited efficiency.

Disclosure of Invention

In order to overcome the problems in the prior art, the invention designs and constructs a series of sTRAIL mutants by using a site-directed mutagenesis technology, and screens out novel mutants, wherein the soluble expression level of the mutant is obviously improved in escherichia coli compared with that of wild type sTRAIL, and the antitumor activity of the mutant is also enhanced to a certain extent. The invention also establishes a preparation method of the sTRAIL mutant by integrating the process steps of induced expression, soluble extraction, separation and purification, processing and refining and the like, and effectively overcomes the problems in the existing sTRIAL expression production and pharmaceutical application.

The specific technical scheme of the invention is as follows:

The mutant of soluble TRAIL related apoptosis inducing ligand contains one or several of R121A, R121S, R121C and V122A mutations, i.e., the mutant is positioned by the full-length amino acid sequence of human TRAIL, the site of the mutation is positioned at the 121 th position or the 122 th position of the full-length amino acid sequence of human TRAIL, the amino acid is mutated from Arg at the 121 th position to Ala, Ser or Cys, and/or from Val at the 122 th position to Ala. The soluble extracellular region of the TRAIL has the same activity of inducing the apoptosis of tumor cells as that of the full-length TRAIL, and the TRAIL is wild type, derived or recombinant TRAIL or the active fragment of the soluble extracellular region thereof or the membrane-bound TRAIL containing the fragment. Preferably, the TRAIL-related apoptosis-inducing ligand is wild-type TRAIL or TRAIL (aa114-281), TRAIL (aa 95-281) and TRAIL (aa 120-281). A preferred mutant of the invention is a mutant of which the amino acid at position 121 in a TRAIL soluble fragment (aa114-281) is mutated from Arg to Ala, which is called delta TRAIL (aa114-281: R121A), the amino acid sequence is shown as SEQ ID No. 1, and the nucleotide sequence is shown as SEQ ID No. 5. Or preferably delta TRAIL (aa 95-281: R121AS, the amino acid sequence is shown as SEQ ID No:2, the nucleotide sequence is shown as SEQ ID No:6, or preferably delta TRAIL (aa 120-281: R121C), the amino acid sequence is shown as SEQ ID No:3, the nucleotide sequence is shown as SEQ ID No:7, or preferably delta TRAIL (aa 120-281: V122A), the amino acid sequence is shown as SEQ ID No:4, and the nucleotide sequence is shown as SEQ ID No: 8.

another objective of the invention is to provide a method for preparing the mutant protein, wherein a fusion gene comprising the coding gene of the mutant of the invention is prepared by artificial synthesis or gene cloning, and is expressed in a corresponding host cell, wherein the host cell comprises Escherichia coli, yeast, insect cell or mammalian cell. A preferred scheme comprises the following steps:

(1) Obtaining a fusion gene comprising the mutant encoding gene;

(2) Cutting the fusion gene obtained in the step (1) and the vector plasmid by using the same restriction enzyme, and then connecting to obtain a recombinant expression plasmid;

(3) Transforming the recombinant expression plasmid obtained in the step (2) into a host cell to form a recombinant cell capable of expressing the mutant protein;

(4) Culturing the recombinant cell obtained in the step (3);

(5) Extracting soluble protein from the cell disruption product, and separating and purifying to obtain the fusion protein of the mutant;

(6) Processing the fusion protein obtained in the step (5) by adopting tobacco etch virus protease;

(7) separating and purifying to obtain the mutant.

in the step (1), the fusion gene containing the mutant coding gene comprises three gene segments (the gene segment at the N-end is a coding gene of a hexahistidine tag, the gene segment at the middle part is a coding gene of a protease cleavage site of the tobacco etch virus, and the gene segment at the C-end is a coding gene of the mutant) and restriction enzyme specific recognition sites located at two ends of the gene.

In the step (2), the vector plasmid is preferably pET-28a (+) plasmid. The restriction enzymes are preferably NcoI and HindIII.

In the step (3), the host cell is preferably Escherichia coli BL21(DE 3).

in the step (4), the recombinant cell is preferably cultured by using IPTG to induce the high-efficiency soluble expression of the fusion protein at a low temperature of 20 ℃.

In the step (5), the mutant fusion protein is extracted, separated and purified. Preferably, the method comprises the steps of suspending the recombinant cells after induction expression in an extracting solution of the non-denatured fusion protein, and obtaining a supernatant solution containing the soluble fusion protein through centrifugal separation after cracking; enriching the fusion protein in the supernatant solution by ammonium sulfate fractional precipitation; redissolving the sediment containing the fusion protein, purifying by using a nickel ion affinity column chromatography, and washing the chromatographic column by using an imidazole concentration gradient solution to obtain an elution peak containing the fusion protein; desalting the elution peak by gel column chromatography and replacing the elution peak with tobacco etch virus protease cutting buffer solution.

In the step (6), the tobacco etch virus protease processes the fusion protein. Preferably, the fusion protein is cleaved with tobacco etch virus protease in a buffer containing 0.1% (v/v) mercaptoethanol for 24h at 4 ℃.

In the step (7), the mutant is separated and purified. Preferably, the mutant protein with the hexa-histidine tag removed is separated from the product obtained after the tobacco etch virus protease cleavage by nickel affinity column chromatography.

The invention also aims to provide application of the mutant in preparing a medicament for treating or preventing diseases related to apoptosis. The mutant transmits a TRAIL death signal to cells through the combination with a death receptor, so that an apoptosis enzyme Caspase system is activated, and finally, the cells are subjected to apoptosis.

furthermore, the medicine can be called an anti-tumor medicine. The medicine contains the mutant of the invention as a medicine active ingredient and a pharmaceutically acceptable carrier.

Furthermore, the medicine also contains other anti-tumor medicines and/or anti-tumor medicine sensitizers.

In one embodiment of the present invention, site-directed mutagenesis, soluble protein analysis and screening for anti-tumor activity were used to demonstrate the effect of N-terminal related amino acid position on soluble expression of sTRAIL and to screen out the mutant Δ TRAIL (aa 114) -281: R121A) (i.e., Arg at position 121 is mutated to Ala) which can significantly improve the efficiency of soluble expression in prokaryotic cells. Compared with wild sTRAIL, the mutant has the advantages that the soluble expression capacity of the mutant in escherichia coli is improved by nearly five times, and the mutant shows improved antitumor activity to a certain extent.

the human tumor necrosis factor related apoptosis inducing ligand mutant can perform soluble expression in Escherichia coli E.coli, and the expression product can be directly obtained by separating and purifying supernatant of E.coli cell wall-broken liquid, so that complicated downstream denaturation and renaturation treatment processes caused by formation of inclusion bodies are avoided.

The invention prepares (from 5 '-end to 3' -end) fusion gene containing hexa-histidine label, tobacco etch virus protease and delta TRAIL (aa 114-. And centrifugally collecting bacterial sludge after induced culture of the engineering bacteria, breaking walls, centrifugally separating supernatant, and purifying by ammonium sulfate fractional precipitation and nickel ion affinity column chromatography to obtain the fusion protein containing the mutant with the purity of more than 95%. After specific cutting by the tobacco etch virus protease, the mutant protein is separated and purified by adopting nickel affinity column chromatography. About 32mg of mutant protein with the purity of more than 95% is obtained in each liter of culture system, and analysis such as N-terminal sequencing and circular dichroism chromatography shows that the mutant has a biological activity structure similar to that of wild type analysis.

₅₀CCK-8 antitumor activity analysis shows that the mutant of the invention shows antitumor activity on human large cell lung cancer NCI-H460 cells, human prostate cancer DU145 cells, human cervical carcinoma Hela cells, human breast cancer MDA-MB-231 cells and MCF-7 cells, wherein the mutant delta TRAIL (aa 114-.

The Annexin-V/PI flow cytometry analysis and Caspase-3 enzyme activity determination show that the anti-tumor function of the mutant is realized by a mechanism of promoting target cell apoptosis and inducing Caspase activity, and the tumor treatment characteristic of a wild type molecule is maintained.

The experimental animal immune identification shows that the immunogenicity of the mutant of the invention is not significantly different from that of a wild type molecule.

Drawings

FIG. 1 shows the construction strategy of pET/His-TRAIL (aa114-281) plasmid containing the gene encoding wild-type TRAIL (aa 114-281).

FIG. 2 shows the site-directed mutagenesis of wild-type TRAIL (aa114-281) and the construction strategy of expression vector.

FIG. 3 shows the analysis and alignment of the DNA sequencing results of site-directed mutant recombinant expression vectors in which the amino acid residues 121, 122, 125 and 126 of TRAIL (aa114-281) are replaced by alanine (Ala, A). Is a site-directed mutant amino acid.

FIG. 4 is the DNA sequencing result analysis and alignment of the site-directed mutant recombinant expression vector in which the 121 th amino acid residue of TRAIL (aa114-281) is replaced by four different amino acid residues. Is site-directed mutant amino acid

FIG. 5 is a schematic diagram of the structure of the fusion protein of wild-type TRAIL (aa114-281) and site-directed mutant. A. Structural mode diagram of wild type TRAIL (aa114-281) fusion protein; structural pattern diagram of mutant fusion protein with alanine substitution at 4 sites (121R, 122V, 125H and 126I) of TRAIL (aa 114-281); structural pattern diagram of mutant fusion protein with R121 of F-I.TRAIL (aa114-281) replaced by 4 amino acid residues (D, S, G and C) at fixed points.

FIG. 6 shows the results of soluble expression studies of wild-type TRAIL (aa114-281) and its mutant fusion protein. Soluble expression SDS-PAGE detection of wild-type TRAIL (aa114-281) and 8 site-directed mutant fusion proteins: t represents total protein of thallus, S represents thallus cracking supernatant, and P represents thallus cracking sediment; C-D, the content detection result of the fusion protein of the soluble wild type TRAIL (aa114-281) and 8 site-directed mutants obtained in each 1L culture system.

FIG. 7 shows the results of the studies on the effect of anti-tumor activity of different site-directed mutagenesis TRAIL (aa114-281) fusion proteins. Expression and isolation and purification of His-delta TRAIL (aa114-281: R121A) fusion protein: m. protein molecular weight standards; 1. inducing total bacterial protein after expression; 2. cell lysis supernatant soluble protein; 3. the total protein is precipitated by 10 to 50 percent of ammonium sulfate of the cracked supernatant; 4. affinity chromatography breakthrough peak; 5-9:40, 60, 80, 100 and 500mM imidazole stage elution peaks. B. The results of the separation and purification of wild-type TRAIL (aa114-281) and site-directed mutant fusion proteins of different amino acid types: m. protein molecular weight standards; 1. a purified wild-type TRAIL (aa114-281) fusion protein, 2. a purified mutant-type Δ TRAIL (aa114-281: R121A) fusion protein, 3. a purified mutant-type Δ TRAIL (aa114-281: V122A) fusion protein, 4. a purified mutant-type Δ TRAIL (aa114-281: R121D) fusion protein, 5. a purified mutant-type Δ TRAIL (aa114-281: R121S) fusion protein, and 6. a purified mutant-type Δ TRAIL (aa114-281: R121C) fusion protein; c. the results of the anti-tumor activity studies of the wild-type TRAIL (aa114-281) and 5 different mutant site-directed mutant fusion proteins.

FIG. 8 shows the preparation and identification of mutant Δ TRAIL (aa114-281: R121A) protein. A. Expression and isolation and purification of mutant delta TRAIL (aa114-281: R121A): m. protein component criteria; 1. total bacterial protein induced for expression; 2. bacteria lysis supernatant; 3. the result of ammonium sulfate fractional precipitation; 4, purifying the result by Ni affinity column chromatography; TEV enzyme specific cleavage results; 6. the recovery result of the target protein delta TRAIL (aa114-281: R121A). B circular dichroism analysis results of mutant delta TRAIL (aa114-281: R121A) and wild-type molecules.

FIG. 9 shows the in vitro anti-tumor activity identification of the fusion protein of wild type and mutant Δ TRAIL (aa114-281: R121A): MDA-MB-231 cells; DU145 cells; c. hela cells; MCF-7 cells; PC3 cells; nci-H460 cells; .

FIG. 10 shows the mechanism of anti-tumor activity of the mutant Δ TRAIL (aa114-281: R121A) protein and fusion protein. a. Apoptosis assay results (Annexin-V/PI double staining); b. determination of the apoptotic enzyme Caspase-3 Activity (spectrophotometry).

Detailed Description

the following examples illustrate specific steps of the present invention, but are not intended to limit the invention.

terms used in the present invention generally have meanings commonly understood by those of ordinary skill in the art, unless otherwise specified. The present invention is described in further detail below with reference to specific examples and with reference to the data. It will be understood that this example is intended to illustrate the invention and not to limit the scope of the invention in any way.

In the following examples, various procedures and methods not described in detail are conventional methods well known in the art.

The present invention is further illustrated by the following specific examples.

Materials, reagents, devices, instruments, apparatuses and the like used in the following examples are commercially available unless otherwise specified.