METHOD FOR PURIFYING AND RENATURATING INCLUSION BODIES OF SCORPION TOXIN PROTEIN AND THEIR USE

Abstract

A method for purifying and renaturating inclusion bodies of scorpion venom protein is provided. The method includes expressing the scorpion venom protein by recombinant Escherichia coli. The C-terminal of the scorpion venom protein has His-tag. The method includes breaking the disulfide bonds in the scorpion venom protein by a denaturating buffer, purifying the denaturated scorpion venom protein with a histidine affinity chromatography column, and renaturating the scorpion venom protein with a renaturation buffer. The renaturation buffer has a pH of 7-9 and includes 50-200 mmol/L Na.sub.2HPO.sub.4, 10-100 mmol/L Tris, 0.1-1 mol/L L-Arg, 1-5 mmol/L EDTA, 0.1-5 mmol/L GSH, 0.05-0.5 mmol/L GSSG, 5-20% (v/v) glycerol, 0.01-5% (v/v) triton X-100. Preparation of the scorpion venom protein by this method has the advantages of simple operation, and good renaturation effect.

Claims

1. A method for purifying and renaturating scorpion venom protein, the method comprising: (1) putting inclusion bodies of scorpion venom protein into a denaturating buffer, stirring, centrifugating, and collecting a precipitate to obtain a denaturated scorpion venom protein; wherein the denaturating buffer has a pH of 7-9 and comprises 50-200 mmol/L Tris, 1-10 mol/L Gdn-HCl, 20-100 mmol/L DTT, and a solvent that is water; (2) purifying the denaturated scorpion venom protein; (3) dissolving the purified denaturated scorpion venom protein in a renaturation buffer, and renaturating the dissolved and purified denaturated scorpion venom protein to obtain a renaturated scorpion venom protein, wherein the renaturation buffer has a pH of 7-9 and comprises 50-200 mmol/L Na.sub.2HPO.sub.4, 10-100 mmol/L Tris, 0.1-1 mol/L L-Arg, 1-5 mmol/L EDTA, 0.1-5 mmol/L GSH, 0.05-0.5 mmol/L GSSG, 5-20% v/v glycerol, 0.01-5% v/v triton X-100, and a solvent that is water.

2. The method for purifying and renaturating scorpion venom protein according to claim 1, wherein the inclusion bodies of the scorpion venom protein are obtained by fermentation of recombinant Escherichia coli.

3. The method for purifying and renaturating scorpion venom protein according to claim 2, wherein the recombinant Escherichia coli is obtained by transforming Escherichia coli by an expression plasmid with cloned scorpion venom protein gene.

4. The method for purifying and renaturating scorpion venom protein according to claim 3, wherein the Escherichia coli is E. coli BL21 (DE3).

5. The method for purifying and renaturating scorpion venom protein according to claim 3, wherein the expression plasmid is pET29a.

6. The method for purifying and renaturating scorpion venom protein according to claim 3, wherein the expression plasmid with cloned scorpion venom protein gene has His-tag at 5-end or 3-end of the scorpion venom protein gene.

7. The method for purifying and renaturating scorpion venom protein according to claim 6, wherein the expression plasmid with cloned scorpion venom protein gene has His-tag at 5-end of the scorpion venom protein gene.

8. The method for purifying and renaturating scorpion venom protein according to claim 1, wherein the denaturated scorpion venom protein is purified with a histidine affinity chromatography column.

9. The method for purifying and renaturating scorpion venom protein according to claim 1, wherein renaturating includes incubating the renaturation buffer and the dissolved and purified denaturated scorpion venom protein at 4-25 C. for 12-72 hours.

10. A scorpion venom protein obtained by the method for purifying and renaturating the scorpion venom protein according to claim 1.

11. An anti-tumor drug preparation, including the scorpion venom protein according to claim 10.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0033] FIG. 1 is a diagram of constructed pET29a-AGAP recombinant plasmid.

[0034] FIG. 2 is result analysis of expression, purification and renaturation of C.sub.His6-rAGAP. Figure A is a SDS-PAGE diagram of C.sub.His6-rAGAP expression; lane 1: whole bacterial protein without IPTG induction; lane 2: whole bacterial protein induced by 1 mmol/L of IPTG at 37 for 4 hours; lane 3: ultrasonic supernatant protein; lane 4, 5: ultrasonic precipitated protein, lane M: protein marker; Figure B is SDS-PAGE diagram of purification and renaturation of C.sub.His6-rAGAP; lane 1: denaturated C.sub.His6-rAGAP protein obtained by nickel column adsorption chromatography purification (diluted 20 fold with a renaturation buffer); lane 2: soluble C.sub.His6-rAGAP protein after renaturation, concentration and centrifugation, lane M: protein marker; Figure C is purification of the renaturated C.sub.His6-rAGAP protein by RP-HPLC (C4 column) analysis.

[0035] FIG. 3 is a diagram of comparing the inhibitory effect on growth of HepG2 tumor cells of C.sub.His6-rAGAP and N.sub.His6-rAGAP obtained by the present invention method obtain with rAGAP expressed by SUMO fusion technology.

[0036] FIG. 4 is inhibitory effect of different doses of C.sub.His6-rAGAP on the tumor size in HepG2 mice model and influence on mice bodyweight. A is a photograph of the tumor taken out of nude mice; B is change of tumor volume in the nude mice determined every three days during drug administration, using 5-FU as a positive control; C is change of nude mice bodyweight determined every three days during drug administration. The statistical difference is analyzed by t-test, * indicates that there is a significant difference between rAGAP or 5-FU drug administration group with model group (p<0.05), ** indicates that there is a significant differencer between AGAP or 5-FU administration group with the model group (p<0.01).

[0037] FIG. 5 is inhibitory effect of C.sub.His6-rAGAP (A) and N.sub.His6-rAGAP (B) on voltage-sensitive sodium current in DRG neuron.

[0038] FIG. 6 is molecular simulations of AGAP, C.sub.His6-rAGAP and N.sub.His6-rAGAP. A is RMSD change of C.sub. atoms of C.sub.His6-rAGAP and N.sub.His6-rAGAP in molecular dynamics simulation (reference model is initial model); B, C is three-dimensional structure of C.sub.His6-rAGAP and N.sub.His6-rAGAP after the dynamic simulation; D, E are three-dimensional electrostatic potential surfaces of AGAP, C.sub.His6-rAGAP and N.sub.His6-rAGAP.

DESCRIPTION OF THE EMBODIMENTS

[0039] Based on the following examples, the present invention can be better understood. However, one skilled in the art will understand that, the content described in the examples is only used to illustrate the invention, and it should not and will not restrict the invention defined in the claims.

Example 1

Construction of Recombinant Plasmid and Strain

[0040] (1) Construction of pET29a/C.sub.His6-rAGAP Plasmid:

[0041] We entrusted GL Biochem (Shanghai) Ltd to synthesize mature peptide sequence of AGAP (this peptide sequence is well known by one skilled in the art), the target gene was amplificated by using P1, P2 primers. The primers had Nde I and Xho I restriction endonuclease recognition site, the target gene was clone into the pET29a plasmid, to obtain pET29a/C.sub.His6-rAGAP recombinant plasmid (FIG. 1). The recombinant plasmid was transferred into E. coli DH5a, after screening in a LB culture medium containing 50 g/ml kanamycin, a DNA sequencing was conducted to determine whether a target gene was correctly introduced into the strain or not. An amplification culture of the strain containing the recombinant plasmid were performed, the recombinant plasmid was extracted, and transferred into a E. coli BL21 (DE3) expression strain.

TABLE-US-00001 P1 (5-GGAATTCCATATGGTACGCGATGGTTATATTGC-3) P2 (5-CCGCTCGAGACCGCCATTGCATTTTCCTG-3)

[0042] (2) Construction of pET43.1/N.sub.His6-rAGAP Plasmid:

[0043] The scorpion toxin target gene was amplificated by using P3, P4 primer, the primers had Nde I and BamH I restriction endonuclease recognition site (marked by an italic underline), P3 primer had His-tag (marked by bold print), and His-tag was introduced into N-terminal of the AGAP, to obtain N.sub.His6-AGAP gene. N.sub.His6-AGAP gene was cloned into pET43.1 plasmid, to obtain pET43.1/N.sub.His6-rAGAP recombinant plasmid. The recombinant plasmid was transferred into E. coli DH5a, after screening in a LB culture medium containing 50 g/ml kanamycin a DNA sequencing was performed, to determine the target gene was whether correctly introduced into the strain. Amplification culture of the strain having the recombinant plasmid was performed, the recombinant plasmid was extracted and transferred into the E. coli BL21 (DE3) expression strain.

TABLE-US-00002 P3 (5-GGAATTCCATATGCATCATCATCATCATCACGTACGCGATGGTT ATATTGC-3) P4 (5-CGCGGATCCTTAACCGCCATTGCATTTTCCTG-3)

Example 2

Expression and Purification of rAGAP

[0044] The E. coli BL21 (DE3) strains having the above recombinant plasmid were respectively cloned into a 50 mL of LB culture medium (containing 50 mg/mL kanamycin), and cultured at 37 at 220 rpm overnight, the next day 25 mL of medium cultured overnight was removed to a 1 L of fresh LB culture medium and cultured to logarithmic growth phase. When absorbance OD.sub.600 reached 0.60.7, 1 mmol/L IPTG was added at 37 and the expression of the target protein was induced for 4 hours. The bacterial liquid was centrifuged, and re-suspended with 100 mL lysis buffer (100 mmol/L NaCl, 50 mmol/L Tris, 2 mmol/L EDTA, 1% v/v tritonX-100, pH 8.0) , and sonicated in an ice bath at 400 w for 30 minutes. The cell lysis buffer was centrifugated at 12000 rpm for 10 minutes. SDS-PAGE founded that almost 90% of rAGAP was expressed in the form of inclusion body (FIG. 2A). The insoluble matter was re-suspended on a 50 mL eluent (100 mmol/L NaCl, 50 mmol/L Tris, 2 mmol/L EDTA, 2 mol/L Urea, pH 8.0), then sonicated for 5 minutes. After the centrifugation, the precipitate was washed with a deionized water for twice, then re-suspended with 10 mL buffer (100 mmol/L Tris, 6 mol/L Gdn-HCl, 50 mmol/L DTT, pH 8.0), mildly stirred for 4 hours, centrifugated at 12000 rpm for 10 minutes, then filtered with a 0.45 m filtration membrane, the insoluble matter was removed, and DTT was removed from the supernatant with a Hi-Prep desalination column, the desalination column was first equilibrate with a buffer A (50 mmol/L Tris, 500 mmol/L NaCl, 8 mol/L urea, pH 8.0). The protein was eluted then applied to the histidine affinity chromatography column equilibrated with the buffer A, and gradient elution were performed successively by using 40, 60, 80, 100, 150, 300 and 500 mmol/L of imidazole eluents (in the eluent, 50 mmol/L Tris, 500 mmol/L NaCl, 8 mol/L urea, pH 8.0 remained unchanged). NaCl and imidazole was eliminated form the eluted rAGAP was with a Hi-Prep desalination column, the desalinating buffer was 50 mmol/L Tris, 8 mol/L urea, pH 8.0. After denaturation and purification, 75 mg of soluble scorpion toxin was obtained in 1 L of culture medium.

Example 3

Renaturation of rAGAP

[0045] The denaturated rAGAP obtained in Example 2 was dissolved in a renaturation buffer (100 mmol/L Na.sub.2HPO.sub.4, 50 mmol/L Tris, pH 8.5, 0.5 mol/L L-Arg, 2 mmol/L EDTA, 1 mmol/L GSH, 0.1 mmol/L GSSG, 5% v/v glycerol, 0.2% v/v triton-100) to a protein final concentration of 0.1 mg/ml, then incubated at 20 for 24 hours. After centrifugation and filtration, the supernatant was concentrated 20 folds by Labscale TFF ultrafiltration system, dialyzed with 1PBS, pH 7.4 buffer for 36 hours, the buffer was changed every 12 hours. After renaturation, 16 mg soluble scorpion toxin can be obtained in 1 L culture medium. The purification of rAGAP was identified by reverse-phase HPLC analysis, the purification may be up to 95% (FIG. 2B, C). The yield and purification in each step of C.sub.His6-rAGAP purification can be seen in Table 1. The key factor in this example was concentration ratio of L-Arg concentration and GSH-GSSG. The method for purifying and renaturating N.sub.His6-rAGAP was same as C.sub.His6-rAGAP.

TABLE-US-00003 TABLE 1 Yield and purity in each step of C.sub.His6-rAGAP purification total weight.sup.# renaturation step (mg) purity* (%) yield (%) total cell extraction 4520 35% washed inclusion 3360 55% 100.00% bodies denaturated protein 594 75% 17.68% nickle column 305 85% 9.10% affinity chromatography renaturated protein 77 85% 2.30% dialysis 65 92% 2.21% .sup.#the weight of AGAP purified from 4 liters of culture medium for Escherichia coli (total wet weight of the cells was 13.41 g) *purity of AGAP was analyzed by Quantity One (Bio-Rad) software

[0046] The key reagents in the renaturation buffer were GSH and GSSG reagents providing redox couple, its concentration optimization process was as follows:

[0047] The concentration of GSH was kept unchanged at 1 mmol/L,

[0048] 1) adjusting pH to 9.0 and final concentration of GSSG to 0.05 mmol/L, and being renaturated at constant temperature of 20 for 24 hours;

[0049] 2) adjusting pH to 9.0 and final concentration of GSSG to 0.1 mmol/L, and being renaturated at constant temperature of 20 for 24 hours;

[0050] 3) adjusting pH to 9.0 and final concentration of GSSG to 0.2 mmol/L, and being renaturatied at constant temperature of 20 for 24 hours;

[0051] 4) adjusting pH to 9.0 and final concentration of GSSG to 0.3 mmol/L, being renaturated at constant temperature of 20 for 24 hours;

[0052] 5) adjusting pH to 9.0 and final concentration of GSSG to 0.4 mmol/L, being renaturated at constant temperature of 20 for 24 hours;

[0053] 6) adjusting pH to 8.5 and final concentration of GSSG to 0.05 mmol/L, being renaturated at constant temperature of 20 for 24 hours;

[0054] 7) adjusting pH to 8.5 and final concentration of GSSG to 0.1 mmol/L, being renaturated at constant temperature of 20 for 24 hours;

[0055] 8) adjusting pH to 8.5 and final concentration of GSSG to 0.2 mmol/L, being renaturated at constant temperature of 20 for 24 hours;

[0056] 9) adjusting pH to 8.5 and final concentration of GSSG to 0.3 mmol/L, being renaturated at constant temperature of 20 for 24 hours;

[0057] 10) adjusting pH to 8.5 and final concentration of GSSG to 0.4 mmol/L, being renaturated at constant temperature of 20 for 24 hours;

[0058] 11) adjusting pH to 8.0 and final concentration of GSSG to 0.05 mmol/L, being renaturated at constant temperature of 20 for 24 hours;

[0059] 12) adjusting pH to 8.0 and final concentration of GSSG to 0.1 mmol/L, being renaturated at constant temperature of 20 for 24 hours;

[0060] 13) adjusting pH to 8.0 and final concentration of GSSG to 0.2 mmol/L, being renaturated at constant temperature of 20 for 24 hours;

[0061] 14) adjusting pH to 8.0 and final concentration of GSSG to 0.3 mmol/L, being renaturated at constant temperature of 20 for 24 hours;

[0062] 15) adjusting pH to 8.0 and final concentration of GSSG to 0.4 mmol/L, being renaturated at constant temperature of 20 for 24 hours;

[0063] 16) adjusting pH to 7.5 and final concentration of GSSG to 0.05 mmol/L, being renaturated at constant temperature of 20 for 24 hours;

[0064] 17) adjusting pH to 7.5 and final concentration of GSSG to 0.1 mmol/L, being renaturated at constant temperature of 20 for 24 hours;

[0065] 18) adjusting pH to 7.5and final concentration of GSSG to 0.2 mmol/L, being renaturated at constant temperature of 20 for 24 hours;

[0066] 19) adjusting pH to 7.5 and final concentration of GSSG to 0.3 mmol/L, being renaturated at constant temperature of 20 for 24 hours s;

[0067] 20) adjusting pH to 7.5 and final concentration of GSSG to 0.4 mmol/L, being renaturated at constant temperature of 20 C. for 24 hours;

[0068] 21) adjusting pH to 7.0 and final concentration of GSSG to 0.05 mmol/L, being renaturated at constant temperature of 20 for 24 hours;

[0069] 22) adjusting pH to 7.0 and final concentration of the GSSG to 0.1 mmol/L, being renaturated at constant temperature of 20 for 24 hours;

[0070] 23) adjusting pH to 7.0 and final concentration of GSSG was 0.2 mmol/L, a being renaturated at constant temperature of 20 for 24 hours;

[0071] 24) adjusting pH to 7.0 and final concentration of GSSG to 0 3 mmol/L, being renaturated at constant temperature of 20 for 24 hours;

[0072] 25) adjusting pH to 7.0 and final concentration of GSSG to 0 4 mmol/L, being renaturated at constant temperature of 20 for 24 hours;

[0073] Base on the above pH and GSSG concentrations, it was found that when pH was 7.58.5 and GSSG concentration was 0.050.2 mmol/L, the renaturation efficiency of rAGAP was higher; wherein, when pH was 8.5 and concentration of GSSG was 0.1 mmol/L, the renaturation efficiency of the rAGAP was highest.

Example 4

Inhibitory Effect of rAGAP on Growth of HepG2 Cells

[0074] The HepG2 in logarithmic growth phase was transferred into a 96-well plate (210.sup.4 cells in each well), and cultured for 24 hours, then added into a serum-free medium and cultured for 12 hours, the cells were washed with a 1PBS, pH 7.4 buffer, then rAGAP of different concentrations were added and incubated for 8 hours. The inhibitory effect of rAGAP on growth of HepG2 cells was determined by MTT method: to each well 10 l MTT (5 mg/ml) was added, and incubated for 4 hours, the medium was removed, and 100 l DMSO was added into each well, shaken at room temperature for 15 minutes, the absorbance was detected by a microplate reader at 570 nm, and inhibition rate of C.sub.His6-rAGAP, N.sub.His6-rAGAP and SUMO-rAGAP against HepG2 cells was calculated (FIG. 3). When concentration of rAGAP was 4 g/ml, inhibition rate of C.sub.His6-rAGAP obtained by present method against HepG2 cells reached 90%, whereas inhibition rate of the SUMO-rAGAP was only 20%, the N.sub.His6-rAGAP almost had no anti-tumor activity in vitro. By calculation, IC.sub.50 of the C.sub.His6-rAGAP against HepG2 cells was 2.5 g/ml, being six fold higher than the SUMO-rAGAP (15 g/ml).

Example 5

Anti-Tumor Activity In Vivo of C.SUB.His6.-rAGAP

[0075] The CD-1 nu/nu mice (thymus removed, bodyweight 1618 g, half male and half female) were purchased from Shanghai Experiment Animal Center of Chinese Academy of Sciences. The mice were subcutaneously injected 510.sup.6 HepG2 cells to make a model, when the tumor volume was grew to about 100 mm.sup.3, the mice were randomized into four groups, 10 mice in each group, the experiment groups were respectively injected 1 mg/kg and 2 mg/kg of C.sub.His6-rAGAP, the positive control group were injected 25 mg/kg of 5-FU, the negative control group was injected with normal saline, the volumes were all 200 l/2 days. The change of tumor volume and bodyweight of the mice were determined every 2 days, after 18 days the mice were killed, and the tumor were taken out. The results demonstrated that, C.sub.His6rAGAP of 2 mg/kg/2 d dose had an obvious inhibitory effect on mice tumor, when the dose was 1 mg/kg/2 d, the inhibitory effect was reduced (FIG. 4A, B). In order to evaluate the influence of C.sub.His6-rAGAP and 5-FU on life quality of the mice, change of mice bodyweight was also determined during the experiment. The results demonstrated that, two groups of different dose C.sub.His6-rAGAP had almost no influence on bodyweight of the mice, whereas bodyweight of the mice in 5-FU group were reduced significantly (FIG. 4C). Furthermore, after 18 days of drug administration, there was no death case in the mice of C.sub.His6-rAGAP administration group, whereas 40% of the mice in 5-FU group were dead.

Example 6

Electrocortical Activity of C.SUB.His6.-rAGAP.

[0076] An adult male C57 mice was killed by CO.sub.2, lumbar (L4, 5 and 6) DRG neuron was taken out, and successively treated with trypsinand collagenaseat 37 for 30 minutes. The cells were mildly sucked and added to a DMEM medium supplemented 10% FBS for culture. The change of the membrane current was recorded with Axon 700A patch-clamp amplifier. In order to selectively record TTX-R sodium current, the composition of external liquid used was 35 mmol/L NaCl, 85 mmol/L choline-Cl, 20 mmol/L TEA-Cl, 3 mmol/L KCl, 1 mmol/L CaCl.sub.2, 1 mmol/L MgCl.sub.2, 10 mmol/L HEPES, 10 mmol/L D-glucose, and the pH was adjusted to 7.4 with Tris. The composition of the internal liquid used was 140 mmol/L CsF, 10 mmol/L NaCl, 10 mmol/L HEPES, 1 mmol/L EGTA, the pH was adjusted to 7.3 with CsOH. The DRG neuron was co-incubated with 500 nmol/L C.sub.His6-rAGAP in a external liquid containing 300 nmol/L TTX for 2 minutes, the recorded TTX-R sodium current was as shown in FIG. 5, C.sub.His6-rAGAP could inhibit about 70% of sodium current.

Example 7

Molecular Simulation of Three-Dimensional Structure of rAGAP

[0077] Using crystal structure of Lqh-alpha-IT (PDB ID: 2ATB) as a template, and AGAP was constructed by using Modeller software, then the molecular dynamics simulation to the three-dimensional structures of C.sub.His6-rAGAP and N.sub.His6-rAGAP were performed for 80 ns in Desmond by using OPLS-AA force field, to investigate the reason of C.sub.His6-rAGAP activity being reserved whereas anti-tumor activity of N.sub.His6-rAGAP being lost. RMSD analysis indicated that in the course of 80 ns of molecular dynamics simulation, His-tag at C-terminal underwent a large structural rearrangement, being close to AGAP surface, whereas the conformational change of His-tag at N-terminal was small, it floated at surface of AGAP (FIG. 6, A, B, C). Protein surface electrostatic potential analysis indicated that, compared with AGAP, influence of His-tag at C-terminal on surface electrostatic potential of C.sub.His6-rAGAP was small, whereas influence of His-tag at N-terminal had a obvious influence on protein surface electrostatic potential (FIG. 6, D, E). Therefore, we infer that loss of N.sub.His6-rAGAP activity may be closely related to change of the charge distribution at AGAP surface, and change of the charge distribution result in change of N.sub.His6-rAGAP surface electrostatic potential.