CN103172727A - Recombinant human proinsulin renaturation and purification method - Google Patents

Abstract

The invention discloses a method for renaturation and simultaneous purification of recombinant human proinsulin by high performance size exclusion chromatography (HPSEC). Including (1) disrupting the bacterial cells containing proinsulin by ultrasonic cell, washing with buffer solution and dissolving in strong denaturing agent (such as 8mol/L or 6-7mol/L guanidine hydrochloride), so as to obtain crudely separated recombinant human insulin (2) Under the optimized chromatographic conditions, the denatured extract containing proinsulin was directly injected, and the purity of the target fraction rhPI obtained by one-step chromatographic elution with a linear gradient of urea concentration can reach more than 96%, and the mass recovery rate is 56.8% %; (3) Further desalting the fraction containing proinsulin, thereby obtaining a fraction with higher purity and activity after renaturation and purification. The recombinant human proinsulin obtained by the method of the present invention can be converted into insulin through enzymatic digestion on a chromatographic column or in a solution, and the chromatographic process is simple to operate, has high repeatability, and is easy for large-scale production.

Description

Renaturation and Purification Method of Recombinant Human Proinsulin

technical field

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

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.

Robert B. M et al [Robert B. M, et al, Protein Expres Purif, 1999, 15: 308-313] used ion exchange chromatography (IEC) and affinity chromatography (AFC) to purify rhPI inclusion body protein, Afterwards, the dilution method was used for renaturation, and the product obtained by enzyme digestion was analyzed by RPLC method. However, Gusarova V et al. [Gusarova V, et al, J Chromatogr A. 2007, 1176: 157-162] reported that the rhPI was renatured by the dilution method first, and then purified by the IEC method and the SEC method respectively. converted to insulin. The above methods all use the dilution method for renaturation, which is difficult to prepare protein drugs on a large scale.

Contents of the invention

The purpose of the present invention is to provide an efficient and rapid method for renaturation and purification of recombinant human proinsulin, which can improve the quality recovery and purity of recombinant human proinsulin, and can be directly used for enzymatic cleavage into insulin, suitable for insulin large-scale production.

The realization process of the present invention is as follows:

The renaturation and purification method of recombinant human proinsulin comprises the following steps:

(1) The bacterial cells of recombinant human proinsulin (rhPI) expressed in Escherichia coli ( E.coli ) were centrifuged, sonicated, washed with buffer I and II respectively, centrifuged and dissolved in strong denaturing buffer to obtain Denatured extract of recombinant human proinsulin;

The buffer I is 10-20 mmol/L PBS, 0.5-1 mmol/L EDTA, pH 7.0-7.5;

The buffer II is 0.5-2.0 mol/L urea, 0.5-1 mmol/L EDTA, 0.5-1.0 mol/L NaCl, 10-20 mmol/L PBS, pH 7.0-7.5;

The strong denaturing buffer is 6.0-8.0 mol/L urea, 10-20 mmol/LDTT, 0.5-1 mmol/LEDTA, pH7.0-8.0; or 6-7 mol/L guanidine hydrochloride, 10-20 mmol/ LDTT, 0.5～1mmol/LEDTA, pH7.0～8.0;

(2) Inject the denatured extract containing recombinant human proinsulin directly into a chromatographic column equilibrated with mobile phase A (20-40 mmol/LPBS, 0-8.0 mol/L urea, pH6.0-7.0), and then use Containing 20-40 mmol/LPBS, pH 6.0-7.0 mobile phase B linear gradient elution, flow rate 0.3-0.7mL/min, collecting target peak chromatographic fractions to obtain renatured and purified chromatographic fractions containing recombinant human proinsulin;

(3) Directly inject the chromatographic fraction containing human proinsulin in step (2), further desalt the chromatographic column equilibrated with mobile phase of 0-10 mmol/LPBS, pH 6.0-7.0, and continue elution for 20-40 min, The flow rate was 0.5-1 mL/min to obtain recombinant human proinsulin.

The chromatographic column in the above step (2) is TSK gel G2000SWXL type (7.8mm×300mm) or Superde×200 10/300GL type (10 mm×300 mm).

The elution method described in the above step (2) can be equilibrated with mobile phase A, followed by linear gradient elution with mobile phase B for 20-40 min, and extended for 5-10 min.

The chromatographic column described in the above step (3) is HiTrap Desalting column.

Advantages of the present invention: the method for refolding and purifying recombinant human proinsulin by using urea concentration gradient size exclusion chromatography provided by the present invention solves the problem of directly diffusing protein molecules from high-concentration denaturants into the mobile phase, and cannot provide a It alleviates the problem of protein refolding environment, and also overcomes the disadvantages of high cost and difficult scale-up of other chromatographic methods. In the present invention, after adding small molecule urea to the mobile phase, a mild environment is provided for protein refolding during the elution process, which promotes protein folding into a compact structure, prolongs retention time, and improves renaturation efficiency and mass recovery rate. get improved. Moreover, it is more conducive to the subsequent enzyme digestion to obtain insulin after passing through the desalting column, the operation is simple, mature and stable, the cost of the stationary phase is low, and it is easy to control and scale up

Description of drawings

Figure 1 SDS-PAGE analysis of rhPI before and after renaturation and purification

Band M: Protein molecular weight standard; Band 1: Uninduced expression of recombinant human proinsulin; Band 2: Induced expression of recombinant human proinsulin for 5 h; Band 3: 8.0 mol/L urea extract; Band 4: Purification and renaturation of rhPI by urea concentration gradient SEC method; Band 5: Secondary purification of desalted fraction; Band 6: Standard insulin; Band 7: Enzymatically converted insulin;

Figure 2 Refolding and purification chromatograms (A ) and mass recovery (B) of rhPI when different urea concentrations were added

In Figure (A), the abscissa is the retention time (time), and the ordinate is the UV absorption value (mAU) at a wavelength of 280nm;

In Figure (B), the abscissa is the concentration of urea added (Urea concentration), and the ordinate is the mass recovery (Mass recovery);

Curve 1: 0.0 mol/L urea; Curve 2: 2.0 mol/L urea; Curve 3: 4.0 mol/L urea; Curve 4: 6.0 mol/L urea; Curve 5: 8.0 mol/L urea;

Figure 3 shows the renaturation and purification of rhPI in different elution modes

The abscissa in the figure is the retention time (time), and the ordinate is the ultraviolet absorption value (mAU) at a wavelength of 280nm;

Curve 1: Add 0.0 mol/L urea to mobile phase A for isocratic elution; Curve 2: Add 2.0 mol/L urea to mobile phase A for isocratic elution; Curve 3: Add 2.0 mol/L urea to mobile phase A L urea gradient elution.

Detailed ways

The technical solutions of the present invention will be described in detail below in conjunction with the embodiments and drawings, but the scope of protection is not limited thereto. The equipment or raw materials used in the examples are all available from the market.

Example 1

(1) Preparation of denatured extract of recombinant human proinsulin

E.coli was used as the host cell to express recombinant human proinsulin (rhPI) protein, and then the fermentation liquid was centrifuged, the cells were broken, 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) washing and centrifugation 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), denatured extract The concentration was 9.92 mg/mL, and the purity of rhPI was above 65.7%, as shown in band 3 in Figure 1 by electrophoresis.

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

HPSEC column (TSK gel G2000SWXL, 300 × 7.8 mm I.D.) was equilibrated with mobile phase A (20 mmol/ LPBS, 4.0 mol/L urea, pH6.5), and 200 μL of denatured extract was injected directly at a flow rate of 0.3 mL/ Perform a linear gradient elution for 30 min at 1 min until the mobile phase B (20 mmol/L PBS, pH 6.5) is 100%, and extend for 10 min. The mass recovery rate of rhPI was 42.5%, and the purity reached over 95%. The chromatogram is shown in curve 3 in Figure 2(A), and the mass recovery rate is shown in Figure 2(B).

(3) Secondary purification of recombinant human proinsulin by desalting chromatographic column

Using a desalting chromatographic column (HiTrap Desalting column) equilibrated with a mobile phase of 0-10 mmol/LPBS, pH 6.0-7.0, directly inject the chromatographic fraction I containing human proinsulin obtained from renaturation and purification, and continue to elute for 30 min, flow rate 1.0ml/min. The purity of rhPI reached more than 99%. See band 5 in Figure 1.

Example 2

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%. The chromatogram is shown in curve 1 of Fig. 2 (A), and the mass recovery rate is shown in Fig. 2 (B).

Example 3

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, pH7.0) 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%. The chromatogram is shown in curve 2 of Fig. 2(A), and the mass recovery rate is shown in Fig. 2(B).

Example 4

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, pH7.0) under the condition of optimizing the urea concentration in the mobile phase and the chromatographic conditions Superde×200 10/300GL SEC column (10×300 mm I.D.), inject 200 μL of the denatured extract directly, and carry 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 10min. The mass recovery rate of rhPI is 51.7%, and the purity can reach more than 96%.

Example 5

Similar to Example 1, the difference is that step (2) uses 100% mobile phase A (20.0 mmol/ LPBS, 2.0 mol/L urea, pH7.5) to equilibrate the TSK gel G2000SWXL HPSEC column (300 × 7.8 mm I.D.), Inject 200 μL of the denatured extract directly, change the flow rate to 0.5 mL/min, and perform isocratic elution with urea concentration or linear gradient elution with urea concentration, respectively, and the mass recovery rate of rhPI in the latter can be increased by 10%. The chromatogram is shown in Fig. 3 curve 2 and curve 3.

Example 6 Proinsulin is converted into insulin

The obtained high-purity recombinant human proinsulin was cleaved with trypsin and carboxypeptidase B for 30 min in a solution at 37°C or under the condition of controlling the column temperature of the HPSEC column at 37°C to convert rhPI into hI. Electrophoresis shows band 7 in Figure 1.

Claims (4)

1. the renaturation of recombinant insulinum primary and purification process is characterized in that comprising the following steps:

(1) the somatic cells of the recombinant insulinum primary of expression in escherichia coli after centrifugal, ultrasonication respectively with damping fluid I and II washing, centrifugally obtain the sex change extract that contains recombinant insulinum primary in being dissolved in strong sex change damping fluid;

Described damping fluid I is 10～20 mmol/L PBS, 0.5～1 mmol/L EDTA, and pH 7.0～7.5;

Described damping fluid II is 0.5～2.0 mol/L urea, 0.5～1 mmol/L EDTA, and 0.5～1.0 mol/L NaCl, 10～20 mmol/L PBS, pH 7.0～7.5;

Described strong sex change damping fluid is 6.0～8.0 mol/L ureas, 10～20mmol/LDTT, 0.5～1mmol/LEDTA, pH7.0～8.0; Or 6～7 mol/L Guanidinium hydrochlorides, 10～20mmol/LDTT, 0.5～1mmol/LEDTA, pH7.0～8.0;

(2) will contain the chromatographic column of the sex change extract direct injection of recombinant insulinum primary to the mobile phase A balance, then with containing 20～40 mmol/LPBS, the Mobile phase B linear gradient elution of pH6.0～7.0, flow velocity is 0.3～0.7mL/min, collects the target peak chromatographic fraction and obtains the chromatographic fraction that renaturation and purifying contain recombinant insulinum primary;

Mobile phase A is: 20～40 mmol/LPBS, 0～8.0 mol/L urea, pH6.0～7.0;

(3) the chromatographic fraction direct injection containing recombinant insulinum primary by step (2), with 0～10 mmol/LPBS, the further desalination of chromatographic column of the moving phase balance of pH6.0～7.0, continue wash-out 20～40 min, flow velocity 0.5～1 mL/min, obtain recombinant insulinum primary.

2. the renaturation of recombinant insulinum primary according to claim 1 and purification process is characterized in that:

In step (2), chromatographic column is TSK gel G2000SWXL type or Superde * 200 10/300GL types.

3. the renaturation of recombinant insulinum primary according to claim 1 and purification process, is characterized in that: after type of elution described in step (2) adopts the mobile phase A balance, with Mobile phase B linear gradient elution 20～40 min, extend 5～10 min.

4. the renaturation of recombinant insulinum primary according to claim 1 and purification process, it is characterized in that: chromatographic column described in step (3) is HiTrap Desalting column.

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