PRODUCTION OF RECOMBINANT HUMAN A-THROMBIN IN PLANT-BASED SYSTEM AND THE APPLICATION THEREOF

Abstract

The present invention relates to a method for producing recombinant human prethrombin-2 protein and having human ?-thrombin activity by the plant-based expression systems.

Claims

1. A method of producing a polypeptide having human ?-thrombin activity in plants, comprising: (1) a gene expression construct comprising a promoter operably linked in the 5-3 direction to a polynucleotide sequence encoding the human prethrombin-2 polypeptide, said gene construct further comprises suitable regulatory elements for plant expression and transformation (2) introducing the gene construct into plant cells, and cultivating said plant cells under suitable conditions, (3) expression, extraction, and purification of the said polypeptide from the plant cells, (4) cleavage of prethrombin-2 polypeptide by protease and obtain active ?-thrombin protein.

2. The method of claim 1, wherein the polynucleotide sequence encoding a human prethrombin-2 polypeptide.

3. The method of claim 1, wherein the gene expression construct comprising an expression promoter that can act in plant, and is selected from a group consisting of constitutive promoters, e.g. CaMV 35S, actin, ubiquitin . . . etc.

4. The method of claim 1, wherein the introducing of the gene construct into plant cells includes infiltrating plant tissues and organs with Agrobacterium cells in the presence of a surfactant, co-cultivation of plant callus with Agrobacterium cells, and particle bombardment-mediated transformation of plant cells, tissues, and organs.

5. The method of claim 1, wherein the plant cells are selected from the group consisting of tobacco, alfalfa, rice, potato, tomato, sunflower, carrot, lettuce, strawberry, moss, duckweed, maize, wheat, and other genetically modifiable plant species.

6. The method of claim 1, wherein the obtained active ?-thrombin is further formulated into pharmaceutical compositions for therapeutic applications, including but not limited to blood clotting disorders or wound healing.

7. The method of claim 2, wherein the polynucleotide sequence was optimized according to the plant codon usage.

8. The method of claim 2, wherein the polynucleotide sequence was fused with an ER retention signal peptide.

9. The method of claim 2, wherein the polynucleotide sequence was fused with a vacuolar retention signal peptide.

10. The method of claim 2, wherein the polynucleotide sequence was fused with a protease cleavage site.

11. The method of claim 10, wherein the protease cleavage site can be recognized by proteases, including but not limited to TEV protease, Enterokinase, SUMO Protease, factor Xa . . . etc.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIG. 1. (A) The gene cassettes of the prethrombin-2 expressed in CaMV 35S and Extin3 terminator (B) The amino acid sequence of prethrombin-2 (MDBprethrombin-2) contains the artificial start codon (methionine, M), recombinant tag for protein purification (8? His tag, HHHHHHHH), vacuole targeting tag for protein trafficking (vacuolar sorting sequence, NPIRL), protease cleavage site for factor Xa (restriction cleavage site, IDGR), and human prethrombin-2. The human prethrombin-2 construct comprises of a light chain and a heavy chain sequence that was maturated and activated by factor Xa digestion. (C) Amino acid sequence alignment of designed construct (MDBprethrombin-2) and wild-type human prethrombin-2 (Hprethrombin-2).

[0019] FIG. 2. Immunoblot analysis displayed that the prethrombin-2 expression level of Agro-infected leaves were harvested and extracted after 3 DPI and 5 DPI (DPI: Day post infiltration). Vacuum infiltration of N. benthamiana leaves with three Agrobacterium tumefaciens strains containing the prethrombin-2 expressing vector, L: LBA4404, E: EHA105, and G: GV3101, respectively. The arrow shows the Prethrombin-2 against anti-his antibody.

[0020] FIG. 3. Immunoblot analysis indicated that the prethrombin-2 was expressed in 6, 7, and 8 weeks of N. benthamiana, respectively. The arrow shows the Prethrombin-2 against anti-his antibody.

[0021] FIG. 4. (A) The 1 L sample of tobacco lysate containing His-tag prethrombin-2 was bound to 5 mL Ni-sepharose 6 fast flow histidine-tagged protein purification resin. Bound material was washed by a linear gradient of up to 20% of B Buffer containing 20 mM sodium phosphate, 500 mM NaCl, and 50 mM imidazole, and the final product was eluted by 250 mM imidazole (black triangle). (B) Immunoblot analysis indicated the purified prethrombin-2 against anti-His antibody. (C) Thrombin activity assay measured at OD.sub.405 nm using S-2238 and the bar chart indicated the relative activity among mock control, factor Xa, and factor Xa+Prethrombin-2.

DETAILED DESCRIPTION

[0022] Human prethrombin-2 was expressed in N. benthamiana plants using viral-based transient expression systems. This invention applies to plant expression cassettes encoding CaMV 35S promoter, human prethrombin-2 and Extin3 terminator (FIG. 1A) in tobacco transient system. The designed construct of human prethrombin-2 named MDBprethrombin-2 which fused sequentially with N-terminal the artificial start codon (Methionine, M), 8?His tag for protein purification, vacuole targeting tag (NPIRL) for protein trafficking, factor Xa (IDGR) of protease cleavage site for formation fragment A and B active form (FIGS. 1B and C). The expression vector containing MDBprethrombin-2 cassette (FIG. 1A) was transformed into three Agrobacterium tumefaciens strains LBA4404, EHA105, and GV3101, respectively. LBA4404-prethrombin-2, EHA105-prethrombin-2, and GV3101-prethrombin-2 were confirmed by PCR and cultured for tobacco infection. Vacuum infiltration of N. benthamian leaves were harvested and extracted after 3 DPI and 5 DPI (DPI: Day post infiltration). Immunoblot analysis of crude plant extracts revealed that all of the LBA4404-prethrombin-2, EHA105-prethrombin-2, and GV3101-prethrombin-2 could be expressed in tobacco transient system and the expression level of 5 DPI was better than 3 DPI (FIG. 2). We also analyzed the growth condition of N. benthamian plants among 6, 7, and 8 weeks and immunoblot analysis of crude plant extracts revealed that the expression level of 8 weeks old plant showed better than 6 and 7 weeks old plant (FIG. 3).

[0023] Purification of human prethrombin-2 from N. benthamian leaves was bound to the affinity chromatography of Ni-sepharose 6 beads fast flow. Bound sample was washed in a linear gradient containing 20 mM sodium phosphate, 500 mM NaCl, and 50 mM imidazole, and the final product was eluted by 500 mM imidazole (FIG. 4A). The purification of prethrombin-2 was achieved FPLC and immunoblot analysis indicated the band of prethrombin-2 against anti-his antibody (FIG. 4B). Thrombin activity assay was measured by S-2238 substrate (H-d-Phe-Pip-Arg-p-nitroanilide, 150 ?M; Chromogenix, Molndal, Sweden). Functional assay of individual sample including of mock control, factor Xa control, and factor Xa+prethrombin-2 were reacted at 37? C. 30 min and measured at OD.sub.405 nm (FIG. 4C).

[0024] Accordingly, the present invention provides a method of making a recombinant prethrombin-2 and fragment thereof that active ?-thrombin form with factor Xa. As we know, full length prothrombin consists of four domains including an N-terminal Gla domain, two kringle sites, and a C-terminal trypsin-like serine protease domain containing 622 a.a. and the M.W of approximately 72 kDa. Prothrombinase is composed of factor Xa, Va, and phospholipids with lower catalytic efficacy on prethrombin-2 than prothrombin. Generally, the ecarin specifically converts prethrombin-2 into active ?-thrombin, is a snake venom-derived protease, this process is an efficient protease to activate thrombin. However, ecarin is not cost-effective and demands extra time and many stages. Hence, many studies aim to address these limitations to reduce the final cost of thrombin production and made this faster with more production yield. In present invention, we expressed the partial prethrombin-2 (295 a.a.) instead of prothrombin (622 a.a) in tobacco and only used the factor Xa to digest the active form of ?-thrombin. FIG. 4C shows The relative thrombin activity was measured at OD.sub.405 containing the mock (1 fold), factor Xa (5.4 fold), and the prethrombin-2+factor Xa (20.1 fold), respectively (FIG. 4C). The data indicates the activity of prethrombin-2+factor Xa could be expressed, purified, and was active in plant system.

EXPERIMENTAL SECTION

[0025] The invention will be further described by means of the following examples which shall not be interpreted as limiting the scope of the appended claims.

Example 1

[0026] MDBprethrombin-2 Construct Design

[0027] The prethrombin-2 construct containing the CAMV (Cauliflower mosaic virus) 35S promoter, partial sequence prothrombin (prethrombin-2), and Extin3 terminator shown in FIG. 1A. The designed prethrombin-2 (MDBprethrombin-2) including of the artificial start codon (methionine, M), recombinant tag for protein purification (8? His tag, HHHHHHHH), vacuole targeting tag for protein trafficking (vacuolar sorting sequence, NPIRL), protease cleavage site for factor Xa (restriction cleavage site, IDGR), and human prethrombin-2 (295 a.a.). The human prethrombin-2 construct comprises of A chain (light chain, ?36-residue) and a B chain (heavy chain, ?259-residue) connecting the endogenous factor Xa digestion. Our construct design indicated the pure MDBprethrombin-2 that was maturated and activated only by factor Xa digestion (FIG. 1C) without the ecarin digestion and one-step purification in Ni-sepharose 6 fast flow histidine-tagged protein purification resin.

Example 2

[0028] Production of Recombinant Prethrombin-2 Using a Viral-Based Transient Expression System in N. benthamiana

[0029] The nucleotide of recombinant prethrombin-2 (MDBprethrombin-2) applied to plant specific codon optimization and confirmed by sequencing. Three agrobacterium strains, LBA4404, EHA105, and GV3101, was grown individually on YEP liquid medium containing 25 ?g/mL Rifampicin at 28? C. overnight. MDBprethrombin-2 was transformed to three agrobacterium strains by freeze-thaw method and grew on YEP solid medium containing 25 g/mL 50 mg of Kanamycin and 25 ?g/mL Rifampicin at 28? C. A single bacterial colony was spread in antibiotics agar plate and checked with colony PCR. The 3 to 5 single bacterial colonies of LBA4404-prethrombin-2, EHA105-prethrombin-2, and GV3101-prethrombin-2 were inoculated into 3 ml of YEP (primary culture) containing the same antibiotics and grown over night on a rotary shaker at 180 rpm at 28? C., respectively. An aliquot of 1 ml bacterial suspension was added to 100 ml YEM medium with the additional antibiotics and grown overnight. Bacteria were pelleted at 5000?g for 5 min and resuspended in 10 mM MgCl.sub.2, 10 mM MES (pH 5.7) with 150 ?M acetosyringone (AS) at a density of 0.1 OD at 600 nm and incubated for 2-3 h at room temperature before vacuum infiltration. The 6-, 7-, 8-old-week of N. benthamiana in each Agrobacterium suspension and were put into a laboratory desiccator connected to a vacuum pump, until the vacuum reached 12 Psi. The 12 Psi vacuum maintained for 1 min followed by a slow release of vacuum and this procedure repeated twice. The infiltration samples were then incubated in a chamber at 25? C. in the light/dark period of 16/8 h. 3 DPI and 5 DPI samples were harvest and extract the total protein by homogenizing leaves with extraction buffer (50 mM Tris-HCl, 300 mM NaCl, 5 mM EGTA, 0.3 mg/ml PMSF). The crude extract was clarified by centrifugation at 14,000?g for 10 min at 4? C.

Example 3

[0030] Purification of Prethrombin-2

[0031] The prethrombin-2 was purified with IMAC (Immobilized Metal Chelate Affinity Chromatography) in AKTA UPC10 (FIG. 4A). The supernatants of plant were bound through the nickel column and washed by 50 mM imidazole. All of the prethrombin-2 was eluted by 250 mM imidazole, and the final buffer replaced in PBS. Immunoblot analysis showed that the purified prethrombin-2 detected against the Anti-His antibody (FIG. 4B).

Example 4

[0032] Functional Assay the Prothrombin-2 to Active ?-Thrombin

[0033] The ?-thrombin was activated by Factor Xa Protease (New England Biolabs), and the specific activity was measured by Chromogenix (Thrombin Substrate S2238). The method for the determination of activity is based on the difference in absorbance (optical density) between the pNA formed and the original substrate. The purified prethrombin-2 was mixed with Factor Xa and 1 mmol/L Chromogenix in PBS. The assays were carried out in 200 ul well in 96 well ELISA plate at 37? C. in assay buffer (50 mM Tris.Math.HCl, 0.15 M NaCl, pH 7.4). After 30 mins, the thrombin activities were determined by spectrum photometer in 405 nm (FIG. 4C).