Recombinant protein renaturation buffer solution as well as preparation method and application thereof

Abstract

The invention relates to a recombinant protein renaturation buffer solution as well as a preparation method and an application thereof. The buffer solution consists of a basis solution, 0.5% of beta-mercaptoethanol, 1% of tween-20, 10mM of beta-cyclodextrin, 1M of L-cysteine and 4M of urea. The preparation method comprises the following steps: firstly, adding 4M of urea into the basis solution, secondly, adding 0.5% of beta-mercaptoethanol, 1% of tween-20, 10mM of beta-cyclodextrin and 1M of L-cysteine according to volume ratio, so as to form the recombinant protein renaturation buffer solution. The buffer solution provided by the invention is applied to recombinant protein renaturation with the primary protein concentration greater than 1mg / mL, is simple to operate, low in cost and convenient in industrial production, the volume after renaturation is slightly increased, the protein concentration is slightly diluted, and the renaturation rate is high.

Description

technical field

[0001] The invention relates to the field of biochemistry, in particular to a recombinant protein refolding buffer and its preparation method and application. Background technique

[0002] The incomplete modification, misfolding and aggregation of the peptide chain due to the expression of the recombinant protein in bacteria generally exists in the form of inclusion bodies. Break the bacteria to obtain pure inclusion bodies. After being dissolved by denaturant (6M guanidine hydrochloride or 8M urea), the inclusion body must be refolded to make the protein fold correctly, improve solubility and restore biological activity before it can reach the level of clinical application. If the protein cannot be renatured well, it will directly manifest as a decrease in solubility and protein precipitation and precipitation, resulting in a large loss of protein. Due to the limitation of technical level, the renaturation and purification of protein is the bottleneck t...

Examples

Embodiment 1

[0011] Example 1 Configuration of Refolding Buffer for Recombinant Protein

[0012] A) Preparation of base solution: add 20mM Tris-HCl and 200mM NaCl to sterile water to adjust the pH to 8.0;

[0013] B) adding 4M urea to the base liquid described in step A;

[0014] C) Add β-mercaptoethanol with a volume ratio of 5‰, 1% Tween-20, 10mM β-cyclodextrin and 1M L-cysteine ​​to the step B solution to form a recombinant protein refolding buffer liquid.

Embodiment 2

[0015] The preparation of embodiment 2 initial protein solution

[0016] Preparation of base fluid:

[0017] Add 20mM Tris-HCl and 200mM NaCl to sterile water to adjust the pH to 8.0;

[0018] Add 8M urea to the base solution, stir evenly, and prepare a mixed solution. Dissolve 10 g of the initially purified recombinant porcine inhibin α subunit protein inclusion body in 500 mL of the mixed solution to obtain a recombinant solution with an initial concentration of 20 mg / mL. Porcine inhibin α subunit protein inclusion body solution (hereinafter referred to as initial protein solution).

[0019] The preliminarily purified recombinant porcine inhibin α subunit protein inclusion body involved in this example was obtained by the applicant according to the method disclosed in Chinese patent CN201010618516.4.

[0020]

Embodiment 3

[0021] Example 3 Refolding of recombinant protein

[0022] Preparation of base fluid:

[0023] Add 20mM Tris-HCl and 200mM NaCl to sterile water to adjust the pH to 8.0;

[0024] (1) Slowly add the initial protein solution obtained in Example 2 with a concentration of 20 mg / mL into the recombinant protein refolding buffer (hereinafter referred to as the buffer) obtained in Example 1 at 4°C, while stirring slowly, the The volume ratio of the initial protein solution to buffer solution is 1:1; after stirring evenly, centrifuge at 12000rpm for 10min at 4°C to collect the protein supernatant;

[0025] (2) Slowly add the base solution into the buffer to form a diluted refolding buffer, in which the final concentration of urea is 1M, and the volume ratio of the base solution to the buffer is 3:1; the step (1) obtained The protein supernatant was slowly added to the diluted buffer solution at 4°C while stirring slowly. The volume ratio of the protein supernatant to the diluted buff...

technical field

[0001] The invention relates to the field of biochemistry, in particular to a recombinant protein refolding buffer and its preparation method and application. Background technique

[0002] The incomplete modification, misfolding and aggregation of the peptide chain due to the expression of the recombinant protein in bacteria generally exists in the form of inclusion bodies. Break the bacteria to obtain pure inclusion bodies. After being dissolved by denaturant (6M guanidine hydrochloride or 8M urea), the inclusion body must be refolded to make the protein fold correctly, improve solubility and restore biological activity before it can reach the level of clinical application. If the protein cannot be renatured well, it will directly manifest as a decrease in solubility and protein precipitation and precipitation, resulting in a large loss of protein. Due to the limitation of technical level, the renaturation and purification of protein is the bottleneck t...

Examples

Embodiment 1

[0011] Example 1 Configuration of Refolding Buffer for Recombinant Protein

[0012] A) Preparation of base solution: add 20mM Tris-HCl and 200mM NaCl to sterile water to adjust the pH to 8.0;

[0013] B) adding 4M urea to the base liquid described in step A;

[0014] C) Add β-mercaptoethanol with a volume ratio of 5‰, 1% Tween-20, 10mM β-cyclodextrin and 1M L-cysteine ​​to the step B solution to form a recombinant protein refolding buffer liquid.

Embodiment 2

[0015] The preparation of embodiment 2 initial protein solution

[0016] Preparation of base fluid:

[0017] Add 20mM Tris-HCl and 200mM NaCl to sterile water to adjust the pH to 8.0;

[0018] Add 8M urea to the base solution, stir evenly, and prepare a mixed solution. Dissolve 10 g of the initially purified recombinant porcine inhibin α subunit protein inclusion body in 500 mL of the mixed solution to obtain a recombinant solution with an initial concentration of 20 mg / mL. Porcine inhibin α subunit protein inclusion body solution (hereinafter referred to as initial protein solution).

[0019] The preliminarily purified recombinant porcine inhibin α subunit protein inclusion body involved in this example was obtained by the applicant according to the method disclosed in Chinese patent CN201010618516.4.

[0020]

Embodiment 3

[0021] Example 3 Refolding of recombinant protein

[0022] Preparation of base fluid:

[0023] Add 20mM Tris-HCl and 200mM NaCl to sterile water to adjust the pH to 8.0;

[0024] (1) Slowly add the initial protein solution obtained in Example 2 with a concentration of 20 mg / mL into the recombinant protein refolding buffer (hereinafter referred to as the buffer) obtained in Example 1 at 4°C, while stirring slowly, the The volume ratio of the initial protein solution to buffer solution is 1:1; after stirring evenly, centrifuge at 12000rpm for 10min at 4°C to collect the protein supernatant;

[0025] (2) Slowly add the base solution into the buffer to form a diluted refolding buffer, in which the final concentration of urea is 1M, and the volume ratio of the base solution to the buffer is 3:1; the step (1) obtained The protein supernatant was slowly added to the diluted buffer solution at 4°C while stirring slowly. The volume ratio of the protein supernatant to the diluted buff...

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