Recombinant human proinsulin renaturation and purification method

A technology of recombinant human insulin and purification methods, applied in the preparation methods of insulin and peptides, chemical instruments and methods, etc., can solve problems such as the preparation of difficult protein drugs, and achieve simple and mature operation, easy control and amplification, and renaturation efficiency. The effect of improving the mass recovery rate

Inactive Publication Date: 2013-06-26
NORTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The above methods all use the dilution method for renaturation

Method used

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  • Recombinant human proinsulin renaturation and purification method
  • Recombinant human proinsulin renaturation and purification method
  • Recombinant human proinsulin renaturation and purification method

Examples

Experimental program
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Embodiment 1

[0028] (1) Preparation of denatured extract of recombinant human proinsulin

[0029] use E. coli As a host cell to express recombinant human proinsulin (rhPI) protein, the fermentation broth was centrifuged, the cells were disrupted, and buffer I (20 mmol / L PBS, 1 mmol / L EDTA, pH 7.4) and II (2.0 mol / L urea, 1 mmol / L EDTA, 1.0 mol / L NaCl, 20 mmol / L PBS, pH 7.4) After washing and centrifuging for crude purification, the obtained inclusion bodies were dissolved in 8.0 mol / L urea, 20 mmol / LDTT , 1.0 mmol / LEDTA, pH 8.0 (or 6-7 mol / L guanidine hydrochloride, 10-20 mmol / LDTT, 0.5-1 mmol / LEDTA, pH 7.0-8.0), the concentration of denatured extract is 9.92 mg / mL, the purity of rhPI is above 65.7%, see electrophoresis figure 1 Middle strip 3.

[0030] (2) Refolding and Purification of Recombinant Human Proinsulin by High Performance Size Exclusion Chromatography

[0031] HPSEC column (TSK gel G2000SWXL, 300 × 7.8 mm I.D.) was equilibrated with mobile phase A (20 mmol / LPBS, 4.0 m...

Embodiment 2

[0035] Similar to Example 1, the difference is that in step (2) equilibrate the TSK gel G2000SWXL HPSEC column (300 × 7.8 mm I.D.) with 100% mobile phase A without urea added, inject 200 μL of the denatured extract directly, and inject 200 μL of the denatured extract at a flow rate of 0.5 Perform linear gradient elution at mL / min for 30 min until the mobile phase B (20.0 mmol / L PBS, pH 6.5) is 100%, and extend for 10 min. The mass recovery rate of rhPI is 39.7%, and the purity can reach more than 95%. Chromatogram see figure 2 (A) Curve 1, mass recovery see figure 2 (B).

Embodiment 3

[0037]Similar to Example 1, the difference is that step (2) is equilibrated with 100% mobile phase A (20.0 mmol / LPBS, 2.0 mol / L urea, pH 7.0) under the condition of optimizing the urea concentration in the mobile phase and the chromatographic conditions TSK gel G2000SWXL type HPSEC column (300 × 7.8 mm I.D.), directly injected 200 μL of denatured extract, and carried out linear gradient elution of urea concentration for 30 min at a flow rate of 0.5 mL / min until mobile phase B (20.0 mmol / L PBS, pH 7.0) was 100%, extended for 10 min. The mass recovery rate of rhPI is 56.8%, and the purity can reach more than 96%. Chromatogram see figure 2 (A) Curve 2, mass recovery see figure 2 (B).

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Abstract

The invention discloses a method for performing high performance size exclusion chromatography (HPSEC) and purifying recombinant human proinsulin renaturation. The method comprises the following steps: (1) performing ultrasonic cell disruption on the proinsulin-containing thallus cell, washing and dissolving the buffer solution in strong denaturant (such as 8mol/L or 6-7 mol/L guanidine hydrochloride) thereby obtaining the recombinant human proinsulin subjected to crude separation; (2) directly feeding the proinsulin-containing denaturated leaching liquor under the optimized chromatographic condition, wherein the purity of target fraction rhPI obtained through one-step chromatography by employing urea concentration linear gradient elute can be over 96 percent, and the mass recovery rate is 56.8 percent; and (3) further desalting the proinsulin-containing original fraction, thereby obtaining the renaturated and purified high-purity and activity fraction. The recombinant human proinsulin obtained by the method can be converted into insulin through digestion on a chromatographic column or digestion in a solution, and the chromatographic process is easy to operate, high in repeatability and easy for large-scale production.

Description

technical field [0001] The invention relates to a method for refolding and purifying recombinant human proinsulin by using high-performance size-exclusion chromatography (HPSEC or SEC), and belongs to the technical field of denatured protein refolding in biomedicine. Background technique [0002] Proinsulin (PI) is a single-chain peptide composed of 86 amino acids, and the two ends of its C peptide are connected to the N-terminal of insulin A chain and the C-terminal of insulin B chain through two basic amino acid residues. The folded coil of the proinsulin molecule ensures the correct docking of the three pairs of disulfide bonds. In the preparation of insulin, it is a mature method to obtain active insulin after direct enzymatic cleavage of proinsulin. Therefore, obtaining active recombinant human proinsulin (abbreviation: rhPI) with high recovery rate is the prerequisite for the preparation of recombinant human insulin. [0003] Robert B. M et al [Robert B. M, et al, P...

Claims

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Application Information

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IPC IPC(8): C07K14/62C07K1/16
Inventor 王骊丽吴溪周慧芳袁洁
Owner NORTHWEST UNIV
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