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Protein refolding method using metal ion chelate affinity chromatography column as solid phase carrier

A technology of metal ions and solid phase carriers, applied in the field of protein renaturation, to achieve the effects of shortening the time of protein renaturation, efficient protein renaturation, and avoiding mutual aggregation

Inactive Publication Date: 2015-08-19
WENZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The application of chromatographic column solid-phase protein renaturation to the protein renaturation of rFGF has not been reported so far

Method used

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  • Protein refolding method using metal ion chelate affinity chromatography column as solid phase carrier
  • Protein refolding method using metal ion chelate affinity chromatography column as solid phase carrier
  • Protein refolding method using metal ion chelate affinity chromatography column as solid phase carrier

Examples

Experimental program
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Effect test

Embodiment 1

[0017] Example 1 Nickel ion metal chelate affinity chromatography column solid-phase protein refolding

[0018] (1) The inclusion body of SUMO-FGF19 was lysed and centrifuged to collect the precipitate, namely SUMO-FGF19;

[0019] (2) Suspend the above precipitate with denaturing buffer (25mM tris(hydroxymethyl)aminomethane-hydrochloric acid buffer (Tris-HCl), pH=8.0, 150mM NaCl, 8M urea, 50mM β-mercaptoethanol), and in Stir at room temperature for 1 hour. Centrifuge at 18,200 rpm for 30 minutes at 4°C, take the supernatant, and detect the protein concentration with the Coomassie Brilliant Blue G250 method;

[0020] (3) Pre-equilibrate the nickel metal ion chelation affinity chromatography column with the above-mentioned denaturing buffer. Load 20 ml of 4 mg / mL denatured SUMO-FGF19 solution on a nickel metal ion chelate affinity column at a flow rate of 1.5 ml per minute; after the loading is complete, wash with denaturing buffer to remove proteins that are not bound to the ...

Embodiment 2

[0021] Example 2 Nickel ion metal chelate affinity chromatography solid-phase protein refolding

[0022] (1) The inclusion body of SUMO-FGF19 was lysed and centrifuged to collect the precipitate, namely SUMO-FGF19;

[0023] (2) Suspend the above precipitate with denaturing buffer (25mM Tris-HCl, pH=8.0, 150mM NaCl, 8M urea, 50mM β-mercaptoethanol), and stir at room temperature for 1 hour. Centrifuge at 18,200 rpm for 30 minutes at 4°C, take the supernatant, and detect the protein concentration with the Coomassie Brilliant Blue G250 method;

[0024](3) Pre-equilibrate the nickel metal ion chelation affinity chromatography column with the above-mentioned denaturing buffer. Load 20 ml of 4 mg / mL denatured SUMO-FGF19 solution on a nickel metal ion chelate affinity column at a flow rate of 1.5 ml per minute; after loading, wash with denaturing buffer to remove unbound protein , until the signal balance; within 12 hours, at a flow rate of 0.1 ml per minute, the mobile phase was r...

Embodiment 3

[0033] Example 3 Cobalt ion metal chelate affinity chromatography solid-phase protein refolding

[0034] (1) The inclusion body of SUMO-FGF19 was lysed and centrifuged to collect the precipitate, namely SUMO-FGF19;

[0035] (2) Suspend the above precipitate with denaturing buffer (25mM Tris-HCl, pH=8.0, 150mM NaCl, 6M guanidine hydrochloride, 50mM β-mercaptoethanol), and stir at room temperature for 1 hour. Centrifuge at 18,200 rpm for 30 minutes at 4°C, take the supernatant, and detect the protein concentration with the Coomassie Brilliant Blue G250 method;

[0036] (3) Pre-equilibrate the cobalt ion metal chelate affinity chromatography column with the above-mentioned denaturing buffer. Load 20 ml of 4 mg / mL denatured SUMO-FGF19 solution on the cobalt ion metal chelate affinity column at a flow rate of 1.5 ml per minute; after the loading is complete, wash with denaturing buffer to remove proteins that are not bound to the column , until the signal is balanced; within 8 ho...

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Abstract

The invention refers to a protein refolding method using metal ion chelate affinity chromatography column as solid phase carrier. The invention uses the specificity affinity relation between rFGF with His or SUMO labels and metal ion chelate affinity chromatography column, attaches the denatured and reduced His-rFGF or SUMO-rFGF to the metal ion chelate affinity chromatography column, replaces denatured buffer solution with refolding buffer solution by gradually changing the composition of mobile phase, so as to prevent the refolding protein folding intermediates from gathering together, and obtain highly effective protein refold while finishing the purification of target protein. Compared to the traditional refolding by dialysis, the method not only can greatly reduce the time needed for protein refolding, but also can make the denatured His-rFGF or SUMO-rFGF obtain refolding protein with high purity and activity in the high concentration(1-10mg / mL), further providing a new process for accomplishing highly effective protein refolding and purifying.

Description

technical field [0001] The invention belongs to a protein refolding method in the field of biopharmaceuticals, and in particular relates to a protein refolding method using a metal ion chelating affinity chromatographic column as a solid phase carrier. Background technique [0002] Fibroblast growth factor (FGF) is a class of peptides with a wide range of biological activities, including 18 members (FGF1-10 and FGF16-23). According to the differences in sequence and structure, FGF can be divided into five paracrine subfamilies (FGF1, FGF3, FGF4, FGF8, and FGF18 subfamilies) and one endocrine subfamily (FGF19 subfamily), which are involved in the development of tissues and organs, blood vessels, etc. Physiological processes such as formation, wound repair, and endocrine regulation. The above biological functions highlight the broad application prospects and promotion value of FGF in the field of clinical medicine, making it a research and development hotspot in the field of ...

Claims

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Application Information

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IPC IPC(8): C07K1/22C07K1/113
Inventor 牛建楼黄志锋周稚晖施璐吴佳敏
Owner WENZHOU MEDICAL UNIV
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