CN114075295A

CN114075295A - Efficient renaturation liquid of Boc-human insulin fusion protein inclusion body and renaturation method thereof

Info

Publication number: CN114075295A
Application number: CN202010839336.2A
Authority: CN
Inventors: 唐亚连; 吴松; 张振山; 杨接运
Original assignee: Suzhou Kunpeng Biotech Co ltd
Current assignee: Suzhou Kunpeng Biotech Co ltd
Priority date: 2020-08-19
Filing date: 2020-08-19
Publication date: 2022-02-22

Abstract

The invention relates to a high-efficiency renaturation solution of a Boc-human insulin fusion protein inclusion body and a renaturation method thereof. Specifically, the invention provides a method for renaturation of Boc-human insulin fusion protein, which comprises the following steps: (1) cracking the inclusion body containing the Boc-human insulin fusion protein to obtain a cracking mixed solution, and adding a denaturing solution into the cracking mixed solution to obtain a Boc-human insulin fusion protein denaturing solution; (2) and mixing the Boc-human insulin fusion protein denaturation solution with a renaturation solution and then reacting to obtain the renatured Boc-human insulin fusion protein. The method of the invention can improve the renaturation efficiency.

Description

Efficient renaturation liquid of Boc-human insulin fusion protein inclusion body and renaturation method thereof

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to a high-efficiency renaturation solution of a Boc-human insulin fusion protein inclusion body and a renaturation method thereof.

Background

Insulin is composed of two amino acid chains of 51 amino acids in total, namely a 21-amino acid A chain and a 30-amino acid B chain, which are connected by two pairs of disulfide bonds between A7-B7 and A20-B19, and an intra-chain disulfide bond between A6-A11 is also arranged in the A chain.

Human insulin is a proteinaceous hormone consisting of 51 amino acids and is secreted by the pancreatic islet cells in the pancreas. Insulin participates in sugar metabolism in the human body, thereby controlling blood sugar balance in the human body. In 1978, the Genetech company in the United states used the first method of biological recombination to express insulin in E.coli. During enzymatic renaturation of "proinsulin", trypsin recognizes lysine at position B29 of insulin, producing a large amount of insulin by-product (DesB 30-insulin) with threonine at position B30 eliminated. Since DesB 30-insulin differs from insulin by only one threonine, the separation between them is very difficult, and therefore, the site-directed introduction of N- (t-butyloxycarbonyl) -lysine (BocK) into the B29 position of recombinant human insulin results in Boc-human insulin fusion protein.

At present, two systems are adopted for producing recombinant human insulin and analogues thereof, one is a yeast system, the yeast system adopts a mode of secreting human proinsulin analogues, and secreted human insulin precursors already have natural disulfide bonds and correct N-ends, but the yeast fermentation production period is long and the expression level is very low. The other is that an escherichia coli system is adopted, but an inclusion body is formed by expression of escherichia coli, and protein is inactive due to mismatching of disulfide bonds between chains and in chains, so that a renaturation process of the inclusion body is required to form correctly paired disulfide bonds.

In the prior art, methods for renaturation of insulin are known, for example, in F-J-rubberlode C07K14/62, chinese patent CN1132845C, which discloses an improved method for obtaining insulin precursors with correctly bonded cystine bonds, i.e. in the presence of cysteine or cysteine hydrochloride and chaotropic auxiliary agents, to obtain products with a correct renaturation of 30% to 50%. Li Wang Li C07K14/62, Chinese patent CN103172727A discloses a method for renaturation by high-efficiency size exclusion chromatography and purification of recombinant human proinsulin, and the result shows that the recovery rate is low when the method is tried in the renaturation of Boc-human insulin fusion protein.

Therefore, there is a need in the art to develop a method for renaturation of Boc-human insulin fusion protein that improves renaturation efficiency.

Disclosure of Invention

The invention aims to provide a renaturation method of Boc-human insulin fusion protein for improving renaturation efficiency.

In a first aspect of the present invention, there is provided a method for renaturation of Boc-human insulin fusion protein, said method comprising the steps of:

(1) cracking the inclusion body containing the Boc-human insulin fusion protein to obtain a cracking mixed solution, and adding a denaturing solution into the cracking mixed solution to obtain a Boc-human insulin fusion protein denaturing solution;

(2) and mixing the Boc-human insulin fusion protein denaturation solution with a renaturation solution and then reacting to obtain the renatured Boc-human insulin fusion protein.

In another preferred embodiment, the inclusion bodies comprise inclusion bodies of recombinant bacteria expressing the Boc-human insulin fusion protein.

In another preferred embodiment, the recombinant bacterium is Escherichia coli.

In another preferred embodiment, the inclusion body is prepared by the following method, which comprises the steps of:

(i) providing a recombinant bacterium for expressing the Boc-human insulin fusion protein;

(ii) and separating the inclusion body containing the Boc-human insulin fusion protein from the recombinant bacteria.

In another preferred embodiment, the amino acid sequence of the Boc-human insulin fusion protein is shown in SEQ ID No. 1, and the 83 th lysine is N epsilon- (tert-butyloxycarbonyl) -lysine.

In another preferred example, in the step (1), the lysis is performed by adding a lysis solution.

In another preferred embodiment, the lysis solution comprises urea.

In another preferred embodiment, the lysis solution comprises 6-10mol/L urea.

In another preferred embodiment, the pH of the lysis solution is in the range of 8.0-10.0, preferably 8.5-9.5.

In another preferred embodiment, the weight/volume ratio (g/ml) of the inclusion body to the lysis solution is 5-40: 200-: 300-600, more preferably 5-25: 350-500.

In another preferred example, in the step (1), the cracking time is 0.5-3 h.

In another preferred example, in the step (1), the temperature of the cracking is room temperature.

In another preferred embodiment, in the step (1), the total protein concentration in the lysis mixture is 10-30g/L, preferably 15-25 g/L.

In another preferred example, in the step (1), the denaturing solution includes a denaturing agent.

In another preferred embodiment, the denaturant is selected from the group consisting of: dithiothreitol, beta-mercaptoethanol, or a combination thereof.

In another preferred embodiment, the concentration of beta-mercaptoethanol is 5 to 50mmol/L, preferably 20 to 30mmol/L, more preferably 25 to 35 mmol/L.

In another preferred embodiment, in the step (1), after adding the denaturing solution to the lysis mixture, the pH is adjusted to 8.0 to 11.0, preferably 9 to 11, and more preferably 9.5 to 10.5.

In another preferred embodiment, the volume ratio of the renaturation solution to the Boc-human insulin fusion protein denaturation solution is 1-10:1, preferably 2-8:1, more preferably 3-7: 1.

In another preferred embodiment, the renaturation solution comprises Tris and Na₂CO₃Glycine, NaCl, EDTA and cystine.

In another preferred example, the solvent of the renaturation solution is water.

In another preferred embodiment, the cystine is L-cystine or D-cystine.

In another preferred embodiment, the concentration of Tris is 20-80mmol/L, preferably 30-70mmol/L, more preferably 40-60mmol/L, most preferably 45-55 mmol/L.

In another preferred embodiment, Na is₂CO₃The concentration of (B) is 1 to 25mmol/L, preferably 3 to 20mmol/L, more preferably 5 to 15mmol/L, most preferably 8 to 12 mmol/L.

In another preferred embodiment, the concentration of glycine is 20-80mmol/L, preferably 30-70mmol/L, more preferably 40-60mmol/L, most preferably 45-55 mmol/L.

In another preferred embodiment, the concentration of NaCl is 20-250mmol/L, preferably 50-150mmol/L, preferably 80-120mmol/L, more preferably 90-110mmol/L, most preferably 95-105 mmol/L.

In another preferred embodiment, the concentration of EDTA is 0.1-5mmol/L, preferably 0.1-2mmol/L, more preferably 0.1-1mmol/L, most preferably 0.3-0.7 mmol/L.

In another preferred embodiment, the concentration of cystine is 0.1-5mmol/L, preferably 0.1-2mmol/L, more preferably 0.1-1.5mmol/L, more preferably 0.1-1mmol/L, most preferably 0.3-0.7 mmol/L.

In another preferred embodiment, the pH of the renaturation solution is 9.0-12, preferably 10-11, more preferably 10.2-10.8, most preferably 10.3-10.7.

In another preferred embodiment, the pH of the renaturation solution is adjusted with an acid or a base.

In another preferred embodiment, in the step (2), the reaction time is 10 to 30 hours, preferably 10 to 25 hours, and more preferably 12 to 18 hours.

In another preferred embodiment, in the step (2), the temperature of the reaction is 0 to 20 ℃, preferably 3 to 15 ℃, and more preferably 2 to 6 ℃.

In another preferred example, in the step (2), the reaction period is 2-100cm³H, preferably 5 to 50cm³H, more preferably 10 to 40cm³H, more preferably 10 to 30cm³H, most preferably 15 to 25cm³The rate of the reaction is such that a gas (e.g., air) is introduced into the reaction system.

In a second aspect of the present invention, a renaturation solution is provided, wherein the renaturation solution comprises Tris, Na₂CO₃Glycine, NaCl, EDTA and cystine.

In another preferred embodiment, the renaturation liquid is as described in the first aspect of the present invention.

The third aspect of the invention provides Boc-human insulin fusion protein, which has an amino acid sequence shown as SEQ ID NO. 1, wherein the 83 th lysine is N epsilon- (tert-butyloxycarbonyl) -lysine.

In a fourth aspect of the invention, there is provided an isolated polynucleotide encoding a Boc-human insulin fusion protein according to the third aspect of the invention.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows a BOC-modified human insulin fusion protein expression plasmid map.

FIG. 2 is a HPLC chart of the sample after renaturation.

FIG. 3 is a HPLC chart of the sample after renaturation.

FIG. 4 shows an electrophoretogram of a renaturation solution sample after renaturation; lane 1: the molecular weight of the standard protein is 100, 30, 25, 20, 15, 10, 5 and 3.4KD from top to bottom in sequence; lane 2: boc-human insulin fusion protein inclusion body denaturation dissolving liquid; lane 3: carrying out reductive detection on the Boc-human insulin fusion protein renaturation solution; lane 4: carrying out non-reducing detection on the Boc-human insulin fusion protein renaturation solution;

FIG. 5 is a HPLC chart of the sample after renaturation.

FIG. 6 is a HPLC chart of the sample after renaturation.

Detailed Description

The present inventors have conducted extensive and intensive studies and have obtained a novel Boc-human insulin fusion protein suitable for expression for the first time. The fusion protein has the advantages of high folding accuracy, high expression rate and high yield, can be folded at high concentration in commercial significance, does not need to purify the recombinant human insulin fusion protein firstly, and directly performs protein renaturation to finally obtain the recombinant human insulin fusion protein with biological activity. The invention also provides a renaturation method and renaturation liquid of the Boc-human insulin fusion protein, and the correctly folded Boc-human insulin fusion protein with high yield can be obtained after the renaturation method and the renaturation liquid are used for renaturating the recombinant human insulin inclusion body denaturation liquid. On this basis, the inventors have completed the present invention.

Term(s) for

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

As used herein, the terms "comprising," "including," and "containing" are used interchangeably and include not only open-ended definitions, but also semi-closed and closed-ended definitions. In other words, the term includes "consisting of … …", "consisting essentially of … …".

As used herein, the term "about" when used in reference to a specifically recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

As used herein, the terms "Tris" and "Tris (hydroxymethyl) aminomethane" are used interchangeably.

As used herein, the terms "EDTA" and "ethylenediaminetetraacetic acid" are used interchangeably.

Unless otherwise indicated, the amino acid sequence of one Boc-human insulin fusion protein of the invention was numbered from N-terminus to C-terminus.

As used herein, the term "BOC-lysine" is used interchangeably with "t-butyloxycarbonyl-lysine".

Fusion proteins

As used herein, "fusion protein of the invention", "recombinant fusion protein", "Boc-human insulin fusion protein", "Boc-modified insulin fusion protein" or "insulin fusion protein" all refer to Boc-human insulin fusion protein having the amino acid sequence shown in SEQ ID No. 1 and the lysine at position 83 is Nepsilon- (t-butyloxycarbonyl) -lysine.

The amino acid sequence shown in SEQ ID NO. 1 is as follows:

MVSKGEELFTGVKLTLKFICTTYVQERTISFKDTYKTRAEVKFEGDENLYFQGRFVNQHLCGSHLVEALYLVCGERGFFYTPKTRGIVEQCCTSICSLYQLENYCN(SEQ ID NO.:1)。

the Boc-human insulin fusion protein can be used for obtaining Boc-human insulin with biological activity after enzyme digestion.

The term "fusion protein" as used herein also includes variants having the above-described activities. These variants include (but are not limited to): deletion, insertion and/or substitution of 1 to 3 (usually 1 to 2, more preferably 1) amino acids, and addition or deletion of one or several (usually up to 3, preferably up to 2, more preferably up to 1) amino acids at the C-terminal and/or N-terminal. For example, in the art, substitutions with amino acids of similar or similar properties will not generally alter the function of the protein. Also, for example, the addition or deletion of one or several amino acids at the C-terminus and/or N-terminus does not generally alter the structure and function of the protein. In addition, the term also includes monomeric and multimeric forms of the polypeptides of the invention. The term also includes linear as well as non-linear polypeptides (e.g., cyclic peptides).

The invention also includes active fragments, derivatives and analogs of the above fusion proteins. As used herein, the terms "fragment," "derivative," and "analog" refer to a polypeptide that substantially retains the function or activity of a fusion protein of the invention. The polypeptide fragment, derivative or analogue of the present invention may be (i) a polypeptide in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide in which a polypeptide is fused with another compound (such as a compound for increasing the half-life of the polypeptide, e.g., polyethylene glycol), or (iv) a polypeptide in which an additional amino acid sequence is fused with the polypeptide sequence (a fusion protein in which a tag sequence such as a leader sequence, a secretory sequence or 6His is fused). Such fragments, derivatives and analogs are within the purview of those skilled in the art in view of the teachings herein.

A preferred class of reactive derivatives refers to polypeptides formed by the replacement of up to 3, preferably up to 2, more preferably up to 1 amino acid with an amino acid of similar or analogous nature compared to the amino acid sequence of the present invention. These conservative variants are preferably produced by amino acid substitutions according to Table A.

TABLE A

The invention also provides analogs of the fusion proteins of the invention. These analogs may differ from the polypeptides of the invention by amino acid sequence differences, by modifications that do not affect the sequence, or by both. Analogs also include analogs having residues other than the natural L-amino acids (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., beta, gamma-amino acids). It is to be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

In addition, modifications may be made to the fusion proteins of the invention. Modified (generally without altering primary structure) forms include: chemically derivatized forms of the polypeptide, such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications in the synthesis and processing of the polypeptide or in further processing steps. Such modification may be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylase. Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides modified to increase their resistance to proteolysis or to optimize solubility.

The term "polynucleotide encoding a fusion protein of the present invention" may include a polynucleotide encoding a fusion protein of the present invention, and may also include polynucleotides that additionally include coding and/or non-coding sequences.

The invention also relates to variants of the above polynucleotides which encode fragments, analogs and derivatives of the polypeptides or fusion proteins having the same amino acid sequence as the present invention. These nucleotide variants include substitution variants, deletion variants and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the fusion protein encoded thereby.

Renaturation liquid

The invention provides a renaturation solution which is used for renaturating a Boc-human insulin fusion protein denaturation solution after the cracking and denaturation of an inclusion body containing Boc-human insulin fusion protein to obtain correctly folded Boc-human insulin fusion protein with high yield. On this basis, the inventors have completed the present invention.

In a preferred embodiment of the present invention, the renaturation solution includes (but is not limited to) Tris, Na₂CO₃Glycine, NaCl, EDTA and cystine.

Specifically, the renaturation liquid is as described in the first aspect of the present invention.

Renaturation method

The renaturation solution is used for carrying out renaturation treatment on the Boc-human insulin fusion protein.

Typically, the present invention provides a method for the renaturation of a Boc-human insulin fusion protein, said method comprising the steps of:

In a preferred embodiment of the present invention, the inclusion body comprises an inclusion body of a recombinant bacterium expressing Boc-human insulin fusion protein.

In another preferred embodiment, the recombinant bacterium is Escherichia coli.

The main advantages of the invention include

1. The invention unexpectedly develops the renaturation liquid and the renaturation method of the Boc-human insulin fusion protein, which can obviously improve the renaturation efficiency.

2. The recombinant human insulin fusion protein with biological activity is finally obtained by directly performing protein renaturation without performing protein purification on the recombinant human insulin fusion protein.

3. The invention greatly improves the renaturation rate to 80-90% by optimizing the renaturation process of the recombinant human insulin inclusion body, is easy to amplify and is suitable for industrial production.

4. The protein expression promoting element of the present invention can improve the solubility of the fusion protein and reduce the intermolecular interaction of the fusion protein, thereby enabling the fusion protein to be folded at a high concentration which is commercially significant.

5. Cyanogen bromide cracking, oxidative sulfitolysis and related purification steps are not required in the process of preparing the target peptide.

6. The preparation of the target peptide does not need to use high-concentration mercaptan or hydrophobic adsorption resin.

7. The peptide of interest is protected from intracellular degradation by the microbial host.

8. The fusion protein can promote the expression of the target peptide, and the expression level and the yield of the target peptide are obviously improved.

9. The fusion protein is very suitable for expressing the target peptide with the unnatural amino acid, and can obviously promote the folding of the target peptide with the unnatural amino acid.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are by weight.

Example 1

1. The recombinant human insulin expression vector is constructed by the methods known in the art, specifically, the descriptions of examples in patent application No. 201910210102.9. The DNA fragment of the fusion protein FP-TEV-R-MiniINS was cloned into the NcoI-XhoI site downstream of the araBAD promoter of the expression vector plasmid pBAD/His A (purchased from NTCC, kanamycin resistance) to obtain the plasmid pBAD-FP-TEV-R-MiniINS. The plasmid map is shown in FIG. 1.

The DNA sequence of pylRs was then cloned into the SpeI-SalI site downstream of the araBAD promoter of the expression vector plasmid pEvol-pBpF (available from NTCC for chloramphenicol resistance), while the DNA sequence of the tRNA (pylTcua) of lysyl-tRNA synthetase was PCR inserted downstream of the proK promoter. This plasmid was designated pEvol-pylRs-pylT.

The plasmid pBAD-FP-TEV-R-MiniINS and the plasmid pEvol-pylRs-pylT are transformed into an escherichia coli strain, and a recombinant escherichia coli strain for expressing the Boc-human insulin fusion protein is obtained through screening. Wherein, the amino acid sequence of the Boc-human insulin fusion protein is shown as SEQ ID NO. 1:

MVSKGEELFTGVKLTLKFICTTYVQERTISFKDTYKTRAEVKFEGDENLYFQGRFVNQHLCGSHLVEALYLVCGERGFFYTPKTRGIVEQCCTSICSLYQLENYCN (SEQ ID NO: 1). Wherein the 83 th lysine is N epsilon- (tert-butyloxycarbonyl) -lysine.

2. Preparing a seed liquid culture medium, inoculating, preparing a second-stage seed liquid through two-stage culture, culturing for 20 hours until the OD600 reaches about 180, obtaining about 3L of fermentation liquid after fermentation is finished, and centrifuging to obtain about 130g/L of wet thalli. And (3) after the fermentation liquor is centrifuged, adding a crushing buffer solution, performing bacteria crushing twice by using a high-pressure homogenizer, adding Tween 80 and EDTA-2Na with a certain concentration after centrifugation, washing once, centrifuging, and collecting precipitates to obtain the inclusion body. Approximately 43g of wet inclusion bodies per liter of fermentation broth are finally obtained.

3. In order to refold the fusion protein, the inclusion bodies are dissolved in 8mol/L urea solution containing 10 to 30mmol/L mercaptoethanol at pH 9.0 to 11.0 so that the concentration of total protein after dissolution is 10 to 30 mg/mL. Diluting the sample by 5-10 times, and carrying out conventional folding for 16-30 hours under the conditions of 4-8 ℃ and pH of 10.5-11.8. And (3) at room temperature, keeping the pH value at 8.0-9.5, and carrying out enzyme digestion on the fusion protein by using trypsin and carboxypeptidase B for 10-20 hours. The reverse phase HPLC analysis result shows that the total yield of the renaturation and enzymolysis steps is higher than 75%. The insulin analogue obtained after enzymatic hydrolysis of trypsin with carboxypeptidase B was designated Boc-human insulin. The sample was clarified by membrane filtration and the Boc-human insulin was initially purified by anion exchange chromatography, with a purity of 90% by SDS-polyacrylamide gel electrophoresis. The final yield of Boc-human insulin per 1 liter of fermentation broth was approximately 2.1 g. And MALDI-TOF mass spectrometry is carried out on the obtained Boc-human insulin, and the result detects that the molecular weight of the Boc-human insulin is consistent with the theoretical molecular weight of 5907.7 Da. Eluting and collecting a sample by ion exchange chromatography, adding hydrochloric acid to perform Boc-human insulin deprotection reaction, adding a sodium hydroxide solution to control the pH to be 2.0-3.2 to terminate the reaction, performing two-step high-pressure reverse phase chromatography, wherein the yield of the purified recombinant human insulin is higher than 75%, and the yield of the finally obtained recombinant human insulin is about 1100mg per 1L of fermentation liquid.

Example 2

This example investigates the renaturation of inclusion bodies of Boc-modified recombinant human insulin fusion protein of example 1 by different renaturation methods as follows:

dissolving 20g of Boc-human insulin fusion protein inclusion body into 500mL of inclusion body dissolving solution containing 8mol/L urea and pH 9.0 at room temperature, and stirring at room temperature for 0.5-3h to make the total protein concentration about 10 g/L; then adding beta-mercaptoethanol with the final concentration of 20mmol/L, and adjusting the pH value to 9.0 to obtain the Boc-human insulin fusion protein denaturation solution.

Adding the Boc-human insulin fusion protein denaturation solution into 5000mL renaturation solution, wherein the final concentration of the renaturation solution is 50mmol/L Tris and 10mmol/L Na₂CO₃50mmol/L glycine, 100mmol/L NaCl, 0.5mmol/L ethylenediaminetetraacetic acid (EDTA), 0.5mmol/L L cystine, pH adjusted to 10.0. At a distance of 20cm³The renaturation solution was continuously aerated at the rate of/H and the reaction was stopped after stirring at 4 ℃ for 16 hours.

HPLC analysis and detection are carried out on the renaturation solution, the HPLC map of a renatured sample is shown in figure 2, the retention time of the HPLC map of the correctly folded and renatured Boc-human insulin fusion protein is 5.733min, the amount of the correctly folded and renatured Boc-human insulin fusion protein actually obtained after the renaturation is calculated according to the peak area normalization method of the Boc-human insulin fusion protein is 2.43g, and the renaturation yield of the Boc-human insulin fusion protein is about 81 percent.

Example 3

dissolving 20g of Boc-human insulin fusion protein inclusion body into 350mL of inclusion body dissolving solution containing 8mol/L urea and pH 9.0 at room temperature, and stirring at room temperature for 0.5-3h to make the total protein concentration about 20 g/L; then adding beta-mercaptoethanol with the final concentration of 30mmol/L, and adjusting the pH value to 10.0 to obtain the Boc-human insulin fusion protein denaturation solution.

Adding the Boc-human insulin fusion protein denaturation solution into 1750mL of renaturation solution, wherein the final concentration of the renaturation solution is 50mmol/L Tris and 10mmol/L Na₂CO₃50mmol/L glycine, 100mmol/L NaCl, 0.5mmol/L EDTA, 0.5mmol/L L cystine, pH adjusted to 10.5. At a distance of 20cm³The renaturation solution was continuously aerated at the rate of/H and the reaction was stopped after stirring at 4 ℃ for 16 hours.

HPLC analysis and detection are carried out on renaturation solution, the HPLC chromatogram of the renaturation solution sample after renaturation is shown in figure 3, and the SDS-PAGE electrophoresis picture is shown in figure 4. As can be seen from FIGS. 3-4, the HPLC chromatogram retention time of the correctly folded and refolded Boc-human insulin fusion protein was 5.749min, the amount of the Boc-human insulin fusion protein actually obtained after refolding calculated according to peak area normalization of the Boc-human insulin fusion protein was 2.55g, and the yield of the refolding of Boc-human insulin fusion protein was calculated to be about 85%.

The insulin fusion protein with the retention time of 5.749min peak-off time is subjected to enzyme digestion by carboxypeptidase and trypsin to obtain BOC-human insulin, and MALDI-TOF mass spectrometry is carried out on the obtained Boc-human insulin, so that the molecular weight of the Boc-human insulin is detected to be consistent with the theoretical molecular weight of 5907.7 Da.

Example 4

dissolving 20g of Boc-human insulin fusion protein inclusion body into 350mL of inclusion body dissolving solution containing 8mol/L urea and pH 9.0 at room temperature, and stirring at room temperature for 0.5-3h to make the total protein concentration about 10 g/L; then adding beta-mercaptoethanol with the final concentration of 20mmol/L, and adjusting the pH value to 10.0 to obtain the Boc-human insulin fusion protein denaturation solution.

Adding the Boc-human insulin fusion protein denaturation solution into 5000mL renaturation solution, wherein the final concentration of the renaturation solution contains 50mmol/L Tris and 10mmol/L Na₂CO₃50mmol/L glycine, 100mmol/L NaCl, 0.5mmol/L EDTA, 2.0mmol/L L cystine, pH adjusted to 10.5. At a distance of 20cm³The renaturation solution was continuously aerated at the rate of/H and the reaction was stopped after stirring at 4 ℃ for 16 hours.

HPLC analysis and detection are carried out on the renaturation solution, the HPLC map of a renatured sample is shown in figure 5, the retention time of the HPLC map of the correctly folded and renatured Boc-human insulin fusion protein is 5.802min, the amount of the correctly folded and renatured Boc-human insulin fusion protein actually obtained after the renaturation is calculated according to the peak area normalization method of the Boc-human insulin fusion protein is 2.34g, and the renaturation yield of the Boc-human insulin fusion protein is calculated to be about 78%.

Example 5

dissolving 20g of Boc-human insulin fusion protein inclusion body into 500mL of inclusion body dissolving solution containing 8mol/L urea and pH 9.0 at room temperature, and stirring at room temperature for 0.5-3h to make the total protein concentration about 10 g/L; then adding beta-mercaptoethanol with the final concentration of 20mmol/L, and adjusting the pH value to 10.0 to obtain the Boc-human insulin fusion protein denaturation solution.

Adding the Boc-human insulin fusion protein denaturation solution into 5000mL renaturation solution, wherein the final concentration of the renaturation solution contains 50mmol/L Tris and 10mmol/L Na₂CO₃50mmol/L glycine, 100mmol/L NaCl, 0.5mmol/L EDTA, 0.5mmol/L L cystine, pH adjusted to 10.0. The reaction was stopped after stirring at 4 ℃ for 16 hours without introducing air.

HPLC analysis and detection are carried out on the renaturation solution, the HPLC map of a renatured sample is shown in figure 6, the retention time of the HPLC map of the correctly folded and renatured Boc-human insulin fusion protein is 5.810min, the amount of the correctly folded and renatured Boc-human insulin fusion protein actually obtained after the renaturation is calculated according to the peak area normalization method of the Boc-human insulin fusion protein is 2.16g, and the renaturation yield of the Boc-human insulin fusion protein is calculated to be about 72%.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

<110> Suzhou spread biotechnology, Inc

<120> high-efficiency renaturation solution of Boc-human insulin fusion protein inclusion body and renaturation method thereof

<130> P2019-1943

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 106

<212> PRT

<213> Artificial Sequence (Artifical Sequence)

<400> 1

Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Lys Leu Thr Leu

1 5 10 15

Lys Phe Ile Cys Thr Thr Tyr Val Gln Glu Arg Thr Ile Ser Phe Lys

20 25 30

Asp Thr Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Glu Asn

35 40 45

Leu Tyr Phe Gln Gly Arg Phe Val Asn Gln His Leu Cys Gly Ser His

50 55 60

Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr

65 70 75 80

Thr Pro Lys Thr Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys

85 90 95

Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asn

100 105

Claims

1. A method for renaturation of Boc-human insulin fusion protein, which is characterized in that the method comprises the following steps:

2. The method of claim 1, wherein the inclusion bodies comprise inclusion bodies of recombinant bacteria expressing the Boc-human insulin fusion protein.

3. The method of claim 1, wherein the Boc-human insulin fusion protein has the amino acid sequence of SEQ ID No. 1 and the 83 th lysine is Nepsilon- (t-butyloxycarbonyl) -lysine.

4. The method of claim 1, wherein the renaturation solution comprises Tris, Na₂CO₃Glycine, NaCl, EDTA and cystine.

5. The method of claim 4, wherein the concentration of Tris is 20-80mmol/L, preferably 30-70mmol/L, more preferably 40-60mmol/L, most preferably 45-55 mmol/L.

6. The method of claim 1, wherein the pH of the renaturation solution is 9.0-12, preferably 10-11, more preferably 10.2-10.8, most preferably 10.3-10.7.

7. The method of claim 1, wherein in step (2), the reaction is carried out at a rate of 2-100cm³H, preferably 5 to 50cm³H, more preferably 10 to 40cm³H, more preferably 10 to 30cm³H, most preferably 15 to 25cm³The rate of the reaction is such that a gas (e.g., air) is introduced into the reaction system.

8. The renaturation liquid is characterized by comprising Tris and Na₂CO₃Glycine, NaCl, EDTA and cystine.

9. The Boc-human insulin fusion protein is characterized by having an amino acid sequence shown as SEQ ID No. 1, wherein the 83 th lysine is N epsilon- (tert-butyloxycarbonyl) -lysine.

10. An isolated polynucleotide encoding the Boc-human insulin fusion protein of claim 9.