Purification and renaturation method of recombinant chimeric protein inclusion body

Abstract

The invention relates to a purification and renaturation method of a recombinant chimeric protein inclusion body, which comprises the following steps: adding arginine into an inclusion body renaturation solution to improve the stability of renatured protein, adding reduced glutathione and oxidized glutathione at the same time, and adjusting the concentration, proportion and other process parameters of the reduced glutathione, the oxidized glutathione and the reduced glutathione to obtain the recombinant chimeric protein inclusion body. The renaturation effect and the yield of the denatured inclusion body protein are further improved.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for purifying and renaturing recombinant chimeric protein inclusion bodies. Background Art

[0002] At present, when the Escherichia coli engineered bacteria constructed by efficient expression plasmids synthesize a large amount of heterologous proteins, they generally exist in the form of insoluble inclusion bodies in bacterial cells. Inclusion bodies are composed of highly aggregated exogenous proteins, nucleic acids, protein synthases and ribosomes. Most of them are expression products of cloned exogenous genes. They have the correct amino acid sequence of the exogenous genes expressed by the engineered bacteria, but the spatial conformation is often wrong. The exogenous proteins in the inclusion bodies have no biological activity and need to be subjected to cell lysis, extraction, washing, denaturation, renaturation and purification steps to obtain soluble and active proteins. If the exogenous protein sequence contains cysteine ​​at the same time, it often forms incorrectly cross-linked disulfide bonds. This easily causes the protein to form insoluble aggregates during the purification and renaturation process. During the purification and renaturation process, the correct pairing of disulfide bonds must be solved. Summary of the invention

[0003] In order to solve the above technical problems, the present invention provides a method for purifying and renaturing recombinant chimeric protein inclusion bodies, wherein the method comprises: (1) Inclusion body preparation: processing the bacterial cells after fermentation culture to obtain inclusion bodies; (2) Dissolving the inclusion bodies: dissolving the inclusion bodies with a denaturing solution to obtain an inclusion body denaturing solution containing the exogenous protein; (3) Protein purification and renaturation, including purification and renaturation of the inclusion body denaturation solution using metal ion chelate affinity chromatography, Wherein, the inclusion body refolding solution contains arginine.

[0004] Preferably, the processing of the fermented bacteria in step (1) includes crushing the bacteria and collecting, washing and centrifuging the precipitate.

[0005] Preferably, the denaturing solution in step (2) comprises 4-10 M urea, 10-100 M Tris, 0-10 mM EDTA, 0-20 mM β-mercaptoethanol, 0-10 mM GSH, and 0-10 mM GSSG.

[0006] Preferably, the inclusion body refolding solution in step (3) contains 0.1-1.0 M L-arginine.

[0007] Preferably, the recombinant chimeric protein includes any recombinant chimeric protein containing a histidine tag, such as a cytokine, an immunogenic protein, and the like. DETAILED DESCRIPTION

[0008] The present invention is further described below in conjunction with specific embodiments, but these embodiments are merely exemplary and do not constitute any limitation to the scope of the present invention.

[0009] Example 1 Preparation of recombinant protein inclusion bodies containing His tags Step 1: Construction and expression of inclusion body vector of target protein The pET-28a(+) recombinant expression plasmid vector containing the target protein encoding gene was prepared according to conventional methods and transformed into Escherichia coli BL21(DE3) strain.

[0010] In the constructed recombinant engineered bacteria, the target gene is placed under the control of the strong promoter T7 promoter. The engineered bacteria can efficiently express the target protein under IPTG induction. When the expression level is high, the expression product is the target protein inclusion body.

[0011] Step 2: Fermentation of engineered bacteria The engineered bacteria glycerol strain was inoculated into a test tube containing 50 μg / ml kanamycin sulfate LB medium at an inoculation ratio of 0.1% (V / V), and cultured at 37°C, 150-200rpm for 14-16 hours to obtain a first-level seed solution; the first-level seed solution was inoculated into a triangular flask containing 50 μg / ml kanamycin sulfate LB medium at an inoculation ratio of 1% (V / V), and cultured at 37°C, 150-200rpm for 3-5 hours to obtain a second-level seed solution; the second-level seed solution was inoculated into a triangular flask containing LB medium at an inoculation ratio of 1% (V / V), and cultured at 37°C, 150-200rpm for 3-5 hours to obtain a third-level seed solution; the third-level seed solution was inoculated into a fermenter (automatic fermenter) at an inoculation ratio of 5-10% (V / V), and the temperature was 37°C, pH The mixture is cultured for 2.5 to 3.5 hours under the conditions of 6.5 to 7.5 and dissolved oxygen above 35%, and then IPTG is added to a final concentration of 0.5 to 1.0 mM, and the culture is continued for 2.5 to 3.5 hours to obtain an engineered bacteria culture solution.

[0012] Step 3: Collection and disruption of bacteria Collect the culture after induced expression in 50 ml centrifuge tubes and centrifuge at 8000 rpm at 4℃ for 10 min. Resuspend the precipitate with an appropriate amount of ultrasonic lysis buffer. [Ultrasonic lysis buffer (20 mmol / L Tris-HCl pH8.0, 0.5 mol / LNaCl, 1 mmol / L EDTA, 1 mg / mL lysozyme)] The resuspended bacterial solution is ultrasonically broken in an ice bath under the following conditions: power of 300W, duty cycle of 50%, 30 s per cycle, and total time of 20 min. The homogenate after breaking is centrifuged at 4℃ and 12000rpm for 15 min. Collect the precipitate, which is the inclusion body.

[0013] Step 4: Processing of inclusion bodies Washing: After the precipitate is resuspended with an appropriate amount of inclusion body washing buffer, stir and wash for 20-30 min, centrifuge at 4°C, 12000 rpm for 15 min, and discard the supernatant. Repeat once. Wash again with an appropriate amount of 50 mmol / L Tris pH8.0 solution (to remove residual EDTA), centrifuge at 4°C, 12000 rpm for 15 min, and discard the supernatant.

[0014] Inclusion body washing buffer (20 mmol / L Tris-HCl pH 8.0, 0.5 mol / L NaCl, 2 mol / L urea, 2% Triton) 2% Triton X-100 solution: Measure 2 ml of Triton X-100 (polyethylene glycol octylphenyl ether) solution and add 98 ml of M buffer.

[0015] Dissolution: Resuspend the precipitate with an appropriate amount of inclusion body dissolution buffer and stir at room temperature for 5-6 hours or overnight. Centrifuge at 12000 rpm and 4℃ for 15 min and collect the supernatant.

[0016] Inclusion body solubilization buffer (20 mmol / L Tris-HCl pH 8.0, 0.5 mol / L NaCl, 8 mol / L urea, 0.2 mmol / L DTT or 100 mmol / L β-mercaptoethanol, 2% Triton) Example 2 Renaturation of inclusion body protein The ratio of inclusion body denaturation solution to renaturation solution (10-50mM Tris, 1.5-2.5mM EDTA, 1.0-10.0% glycerol, 1.5-2.5mM DTT, 0.1-1.0M L-arginine, pH 9.00) is 1:150-300 (V / V), the addition flow rate is 0.6ml / min, and a magnetic stirrer is used for stirring during the addition. After the addition is completed, the stirring is stopped and the mixture is allowed to stand at 2-8°C for 24-72h to obtain a protein renaturation solution.

[0017] Example 3 Purification of inclusion body protein The ion exchange chromatography medium used was Q SepharoseTM High Performance; the ion exchange chromatography liquid A was used to balance the chromatography column, the linear flow rate was 24 cm / h, and the sample was loaded after the UV detection baseline was stable. After loading, the chromatography column was balanced with ion exchange chromatography liquid A (15-25 mM Tris, pH 7.5), and ion exchange chromatography liquid B (15-25 mM Tris, 55-65 mM NaCl, pH 7.5) was used for isocratic elution, and the target peaks produced in this stage were collected.

Claims

1. A method for renaturation and purification of recombinant chimeric protein inclusion bodies, characterized in that: The method includes: (1) Processing the fermented bacterial cells to obtain inclusion bodies; (2) dissolving the inclusion bodies, dissolving the inclusion bodies with a denaturing solution to obtain an inclusion body denatured solution containing cleaved proteins; (3) Protein renaturation: adding the inclusion body denaturation solution dropwise to the inclusion body renaturation solution to dilute and renature the protein to obtain a protein renaturation solution; (4) Protein purification, including purification of the protein refolding solution by ion exchange chromatography.

2. The method according to claim 1, characterized in that The processing of the fermented bacteria in step (1) includes breaking the bacteria, collecting the precipitate, washing and centrifuging.

3. The method according to any one of claims 1-2, characterized in that: The denaturing solution in step (2) includes 10-50 mmol / L Tris, 0.1-0.5 mol / L NaCl, 4.0-10.0 mol / L urea, 0-100 mmol / L β-mercaptoethanol, and 2% Triton.

4. The method according to any one of claims 1 to 3, characterized in that: The inclusion body refolding solution in step (3) contains 0-1.0 mol / L L-arginine.