TCEP-based recombinant proinsulin inclusion body denaturation and renaturation method

Abstract

The invention relates to a TCEP-based recombinant proinsulin inclusion body denaturation and renaturation method, according to the method, TCEP is added as a reducing agent in the denaturation and renaturation process, compared with the prior art, the yield of proinsulin denaturation and renaturation is increased, and the yield of insulin is remarkably increased. Besides, TCEP is high in reducibility, wide in applicable pH range and extremely low in toxicity, so that the production efficiency and the process stability of the recombinant insulin production process can be remarkably improved, the harm to operators and the environment is reduced, and the requirements of green chemistry and safe production are met.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for the renaturation of recombinant proinsulin inclusion bodies based on TCEP. Background Technology

[0002] Insulin, a small protein containing three disulfide bonds, is highly dependent on the correct pairing of these bonds for its biological activity. Yeast and *E. coli* are two commonly used expression systems in the production of recombinant insulin. The yeast system has the advantage of achieving soluble expression of the target protein, simplifying downstream processes, but its fermentation yield is typically low. Therefore, the *E. coli* system, with its higher fermentation yield, is more commonly used in industrial production. However, in this system, the recombinant insulin expression product often exists in the form of inclusion bodies. The proteins in these inclusion bodies are usually misfolded or unfolded, and the molecules are tightly aggregated together through non-covalent interactions and intermolecular disulfide bonds. An effective method is needed to depolymerize them into soluble monomers and then perform subsequent refolding. Existing methods for denaturing recombinant insulin include: (1) Traditional reducing agents include β-mercaptoethanol and dithiothreitol (DTT), but the reducing agents used in traditional processes have obvious limitations: β-mercaptoethanol has strong toxicity and poses a threat to the health of operators during the production process. At the same time, it is highly volatile and produces a pungent odor, which pollutes the working environment. Although DTT has relatively low toxicity, it is expensive and has poor stability under certain reaction conditions, resulting in low reaction efficiency and affecting the overall effect of insulin renaturation.

[0003] (2) Patent CN1163600C describes the reversible sulfonation of sulfhydryl groups in insulin by adding sodium tetrathionate and sodium sulfite. However, the use of this method is limited due to the high toxicity of sodium tetrathionate, which harms human health and the environment. Currently, there are no patent reports on the application of highly efficient, low-toxicity, and stable reducing agents in insulin purification processes. Summary of the Invention

[0004] To address the aforementioned technical problems in the prior art, this invention provides a TCEP-based method for the renaturation of recombinant proinsulin inclusion bodies.

[0005] A TCEP-based denaturation and renaturation method involves denaturing and dissolving collected recombinant proinsulin inclusion bodies using denaturation buffer, adding TCEP hydrochloride during or after the denaturation and dissolution of the inclusion bodies, and reducing disulfide bonds at pH 1.5–9. The solution is diluted and refolded; after refolding, it is oxidized, and the oxidized solution is concentrated to obtain recombinant proinsulin.

[0006] Furthermore, the denaturing buffer is 4-8 mol / L urea or 4-6 mol / L guanidine hydrochloride.

[0007] Furthermore, the amount of TCEP hydrochloride added is 0.5~50 mmol / L to a final concentration.

[0008] Furthermore, TCEP hydrochloride is added in solid or aqueous solution form.

[0009] Furthermore, the oxidation is performed by adding an oxidizing agent or by oxidation in air.

[0010] Furthermore, the oxidant is a copper sulfate solution.

[0011] Furthermore, recombinant proinsulin refers to recombinant proinsulin expressed in Escherichia coli using gene recombination technology.

[0012] Furthermore, the recombinant proinsulin is selected from insulin icotin, insulin degludec, insulin glargine, human insulin, and insulin lispro.

[0013] Furthermore, the dilution and refolding process involves adding inclusion body lysate to a refolding buffer containing 20-50 mmol / L potassium carbonate at a final protein concentration of 0.1-1.0 mg / mL, adding 0.5-5 g L-cysteine ​​hydrochloride per g of protein, and diluting and refolding at 2℃-16℃ for 12-16 h.

[0014] Furthermore, the concentration is a 3 kDa ultrafiltration concentration.

[0015] The TCEP hydrochloride mentioned in this invention refers to (Tris(2-carboxyethyl)phosphine hydrochloride, TCEP·HCl, CAS 51805-45-9). Attached Figure Description

[0016] Figure 1 HPLC chromatogram of the inclusion body denaturation and reduction sample in Example 2.

[0017] Figure 2 HPLC chromatogram of the re-oxidized sample in Example 2.

[0018] Figure 3 HPLC chromatogram of the 3kDa ultrafiltration sample in Example 2.

[0019] Figure 4 HPLC chromatogram of the inclusion body after denaturation and reduction in Example 3.

[0020] Figure 5HPLC chromatogram of the re-oxidized sample in Example 3.

[0021] Figure 6 HPLC chromatogram of the 3kDa ultrafiltration sample in Example 3. Detailed Implementation

[0022] The specific embodiments of the present invention will be described in more detail below with reference to the accompanying drawings and examples, so as to better understand the solution of the present invention and its advantages in various aspects. However, the specific embodiments and examples described below are for illustrative purposes only and are not intended to limit the present invention.

[0023] The following uses Ico insulin as an example to illustrate the renaturation method of recombinant proinsulin inclusion bodies based on TCEP. Example 1

[0024] The construction method of the proinsulin-expressing strain is as follows: [The text abruptly ends here, likely due to an incomplete sentence or missing information.] Nde I / Xho I The pET-27b(+) plasmid containing the proinsulin gene inserted at the site was transformed into E. coli to obtain a proinsulin expression strain, which was then induced to produce proinsulin through fermentation. Example 2

[0025] 1) Inclusion body collection: Take 1 kg of E. coli cells collected by centrifugation during fermentation, suspend them in TE buffer, homogenize the suspension twice using a homogenizer at a pressure of approximately 850 bar, and collect inclusion bodies after centrifugation. Wash twice with TE buffer and collect the washed inclusion bodies.

[0026] 2) Inclusion body denaturation and reduction: This embodiment involves adding solid TCEP hydrochloride during the denaturation and dissolution of inclusion bodies. Specifically, after washing, 1 g of inclusion body was added to 10 mL of 8 mol / L urea containing 10 mmol / L TCEP hydrochloride, and the mixture was stirred and dissolved at pH 6.8 for 8 h. After denaturation and reduction, the sample was analyzed by HPLC at 214 nm using a C8 column (4.6 mm × 150 mm). The RP-HPLC detection conditions were: mobile phase A: 10% acetonitrile + 100 mM sodium dihydrogen phosphate, pH 7.0; mobile phase B: 50% acetonitrile; elution gradient: 30%~80% B for 50 min, 80%~30% B for 1 min, 30% B for 9 min; flow rate 1 mL / min. The results are as follows: Figure 1 As shown in the figure, the RP-HPLC purity of the reduced sample was 50.0%.

[0027] 3) Renaturation and oxidation: Inclusion body lysate was added to a refolding buffer containing 30 mmol / L potassium carbonate at a protein concentration of 0.5 mg / mL. 2 g of L-cysteine ​​hydrochloride was added per g of protein, and the mixture was refolded at 12°C for 12 h. The refolded sample was then oxidized with copper sulfate solution to a final concentration of 10 μmol / L for 4 h to obtain 23.5 g of proinsulin with a RP-HPLC purity of 53.7%. Figure 2 ) 4) 3 kDa ultrafiltration: The oxidized solution was concentrated by 3 kDa ultrafiltration, and the solution system was then replaced to obtain 21.9 g of proinsulin with a purity of 55.2% by RP-HPLC. Figure 3 ) Example 3

[0028] 1) Inclusion body collection: Take 1 kg of E. coli cells collected by centrifugation during fermentation, suspend them in TE buffer, homogenize the suspension twice using a homogenizer at a pressure of approximately 850 bar, and collect inclusion bodies after centrifugation. Wash twice with TE buffer and collect the washed inclusion bodies.

[0029] 2) Inclusion body dissolution and denaturation: This embodiment involves adding TCEP hydrochloride aqueous solution after the inclusion bodies have denatured and dissolved. Specifically, after washing, 1 g of inclusion body was added to 8 mL of 8 mol / L urea, and the mixture was stirred and dissolved for 6 h. Then, 1 / 100 volume of 200 mmol / L TCEP hydrochloride aqueous solution was added (to make the final concentration of TCEP hydrochloride in the system 2 mmol / L), and the reaction was carried out at pH 8.0 for 1 h. After the inclusion bodies were denatured and reduced, the sample was analyzed by HPLC using a C8 column (4.6 mm × 150 mm) at 214 nm. The RP-HPLC detection conditions were: mobile phase A: 10% acetonitrile + 100 mM sodium dihydrogen phosphate, pH 7.0; mobile phase B: 50% acetonitrile; elution gradient: 30%~80% B for 50 min, 80%~30% B for 1 min, 30% B for 9 min; flow rate: 1 mL / min. The results are as follows: Figure 4 As shown in the figure, the RP-HPLC purity of the reduced sample was 50.1%.

[0030] 3) Renaturation and oxidation: Inclusion body lysate was added to a refolding buffer containing 30 mmol / L potassium carbonate to achieve a final protein concentration of 0.2 mg / mL. 3 g of L-cysteine ​​hydrochloride was added per g of protein, and the mixture was refolded at 8°C for 16 h. The refolded sample was then oxidized with copper sulfate (final concentration 10 μmol / L) for 6 h to obtain 22.2 g of proinsulin with a RP-HPLC purity of 57.1%. Figure 5) 4) 3kDa ultrafiltration: The oxidized solution was concentrated by 3 kDa ultrafiltration, and the solution system was then replaced to obtain 20.8 g of proinsulin with a purity of 60.9% by RP-HPLC. Figure 6 ) The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A variable complexity method based on TCEP, characterized in that, The collected recombinant proinsulin inclusion bodies were denatured and dissolved using denaturing buffer. TCEP hydrochloride was added during or after the denaturation and dissolution of the inclusion bodies to reduce disulfide bonds at pH 1.5–9. Perform dilution and refolding; After refolding, oxidation is performed, and the oxidized solution is concentrated to obtain recombinant proinsulin.

2. The method according to claim 1, characterized in that, The denaturing buffer solution is 4-8 mol / L urea or 4-6 mol / L guanidine hydrochloride.

3. The method according to claim 1, characterized in that, The amount of TCEP hydrochloride added is 0.5~50 mmol / L to a final concentration.

4. The method according to claim 1, characterized in that, TCEP hydrochloride is added in solid or aqueous solution form.

5. The method according to claim 1, characterized in that, The oxidation is performed by adding an oxidizing agent or by oxidation in air.

6. The method according to claim 1, characterized in that, The oxidant is a copper sulfate solution.

7. The method according to claim 1, characterized in that, Recombinant proinsulin refers to recombinant proinsulin expressed in Escherichia coli using gene recombination technology.

8. The method according to claim 1, characterized in that, The recombinant proinsulin is selected from insulin icotin, insulin degludec, insulin glargine, human insulin, and insulin lispro.

9. The method according to claim 1, characterized in that, The dilution and refolding process involves adding inclusion body lysate to a refolding buffer containing 20-50 mmol / L potassium carbonate at a final protein concentration of 0.1-1.0 mg / mL, adding 0.5-5 g L-cysteine ​​hydrochloride per g of protein, and diluting and refolding at 2℃-16℃ for 12-16 h.

10. The method according to claim 1, characterized in that, The concentration is a 3 kDa ultrafiltration concentration.

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