CN114075295B - Efficient renaturation solution of Boc-human insulin fusion protein inclusion body and renaturation method thereof - Google Patents

Efficient renaturation solution of Boc-human insulin fusion protein inclusion body and renaturation method thereof Download PDF

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CN114075295B
CN114075295B CN202010839336.2A CN202010839336A CN114075295B CN 114075295 B CN114075295 B CN 114075295B CN 202010839336 A CN202010839336 A CN 202010839336A CN 114075295 B CN114075295 B CN 114075295B
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boc
fusion protein
solution
human insulin
renaturation
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CN114075295A (en
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唐亚连
吴松
张振山
杨接运
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Suzhou Kunpeng Biotech Co ltd
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/62—Insulins
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Abstract

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

Description

Efficient renaturation solution of Boc-human insulin fusion protein inclusion body and renaturation method thereof
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to a high-efficiency renaturation solution of Boc-human insulin fusion protein inclusion bodies and a renaturation method thereof.
Background
Insulin is a 51 amino acid composition of two amino acid chains, 21 amino acid A chain and 30 amino acid B chain, connected by two pairs of disulfide bonds between A7-B7 and A20-B19, and one A6-A11 intra-chain disulfide bond within the A chain.
Human insulin is a protein hormone consisting of 51 amino acids secreted by islet cells within the pancreas. Insulin participates in the metabolism of sugar in the human body, thereby controlling the blood glucose balance in the human body. In 1978, genetech in the united states used the method of biological recombination to express insulin in escherichia coli for the first time. During cleavage renaturation of "proinsulin", trypsin recognizes lysine at position B29 of insulin, producing a large amount of threonine-depleted insulin by-product at position B30 (DesB 30-insulin). Since DesB 30-insulin differs from insulin by only one threonine, separation between them is very difficult, and thus, the introduction of N- (tertiary Ding Yangtan-yl) -lysine (BocK) site-directed to 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. And secondly, an escherichia coli system is adopted, but escherichia coli expression forms inclusion bodies, disulfide bond mismatch between chains and in chains causes protein inactivity, and a renaturation process of the inclusion bodies is required to form disulfide bonds which are correctly paired.
In the prior art, related to methods of renaturation of insulin, for example, in F-J Lu Baluo De C07K14/62, china patent CN1132845C, an improved method of obtaining insulin precursors with correctly bonded cystine bonds is disclosed, i.e., obtaining 30% to 50% of the correctly renatured product in the presence of cysteine or cysteine hydrochloride and a chaotropic auxiliary agent. Wangli C07K14/62, china patent CN103172727A discloses a method for renaturation by high-efficiency size exclusion chromatography and purification of recombinant human proinsulin, and the method is tried to renaturate Boc-human insulin fusion protein, and the result shows that the recovery rate is low.
Accordingly, there is a need in the art to develop a renaturation method of Boc-human insulin fusion proteins that increases 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 of renaturation of a Boc-human insulin fusion protein, said method comprising the steps of:
(1) Cracking inclusion bodies containing the Boc-human insulin fusion protein to obtain a cracking mixed solution, and adding a denaturation solution into the cracking mixed solution to obtain a Boc-human insulin fusion protein denaturation solution;
(2) And mixing the modified solution of the Boc-human insulin fusion protein with renaturation solution, and reacting to obtain the renaturated 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 E.coli.
In another preferred embodiment, the inclusion bodies are prepared by a method comprising the steps of:
(i) Providing a recombinant bacterium expressing a Boc-human insulin fusion protein;
(ii) And separating inclusion bodies 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 lysine 83 is N [ epsilon ] -t-butoxycarbonyl) -lysine.
In another preferred embodiment, in the step (1), the lysis is performed by adding a lysis solution.
In another preferred embodiment, the lysate comprises urea.
In another preferred embodiment, the lysate comprises 6-10mol/L urea.
In another preferred embodiment, the pH of the lysate is 8.0-10.0, preferably 8.5-9.5.
In another preferred embodiment, the weight to volume ratio (g/ml) of the inclusion bodies to the lysate is 5-40:200-800, preferably 10-30:300-600, more preferably 5-25:350-500.
In another preferred embodiment, in the step (1), the time of the cleavage is 0.5 to 3 hours.
In another preferred embodiment, in the step (1), the temperature of the cleavage is room temperature.
In another preferred embodiment, in the step (1), the total protein concentration in the lysis mixture is 10-30g/L, preferably 15-25g/L.
In another preferred embodiment, 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 combinations thereof.
In another preferred embodiment, the concentration of beta-mercaptoethanol is from 5 to 50mmol/L, preferably from 20 to 30mmol/L, more preferably from 25 to 35mmol/L.
In another preferred embodiment, in step (1), after adding the denaturing solution to the lysis mixture, the pH is adjusted to 8.0-11.0, preferably 9-11, more preferably 9.5-10.5.
In another preferred embodiment, the volume ratio of renaturation solution to the Boc-human insulin fusion protein denaturing solution is 1-10:1, preferably 2-8:1, more preferably 3-7:1.
In another preferred embodiment, the renaturation solution comprises Tris, na 2CO3, glycine, naCl, EDTA and cystine.
In another preferred embodiment, 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 45-55mmol/L.
In another preferred embodiment, the Na 2CO3 is present in a concentration of 1 to 25mmol/L, preferably 3 to 20mmol/L, more preferably 5 to 15mmol/L, most preferably 8 to 12mmol/L.
In another preferred embodiment, the glycine concentration is 20-80mmol/L, preferably 30-70mmol/L, more preferably 40-60mmol/L, most preferably 45-55mmol/L.
In another preferred embodiment, the concentration of NaCl is 20-250mmol/L, preferably 50-150mmol/L, more preferably 80-120mmol/L, still more preferably 90-110mmol/L, most preferably 95-105mmol/L.
In another preferred embodiment, the EDTA concentration is 0.1-5mmol/L, preferably 0.1-2mmol/L, more preferably 0.1-1mmol/L, most preferably 0.3-0.7mmol/L.
In another preferred embodiment, the cystine concentration 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 0.3-0.7mmol/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 step (2), the reaction time is 10 to 30 hours, preferably 10 to 25 hours, more preferably 12 to 18 hours.
In another preferred embodiment, in step (2), the temperature of the reaction is 0-20 ℃, preferably 3-15 ℃, more preferably 2-6 ℃.
In another preferred embodiment, in step (2), a gas (e.g., air) is introduced into the reaction system during the reaction at a rate of 2-100cm 3/H, preferably 5-50cm 3/H, more preferably 10-40cm 3/H, more preferably 10-30cm 3/H, and most preferably 15-25cm 3/H.
In a second aspect of the present invention, there is provided a renaturation solution comprising Tris, na 2CO3, glycine, naCl, EDTA and cystine.
In another preferred embodiment, the renaturation solution is as described in the first aspect of the present invention.
In a third aspect of the invention, there is provided a Boc-human insulin fusion protein having an amino acid sequence as shown in SEQ ID NO. 1 and lysine at position 83 is N [ epsilon- (t-butoxycarbonyl) -lysine.
In a fourth aspect of the invention there is provided an isolated polynucleotide, wherein said polynucleotide encodes a Boc-human insulin fusion protein according to the third aspect of the invention.
It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
Drawings
FIG. 1 shows a map of the expression plasmid of the BOC-modified human insulin fusion protein.
FIG. 2 is a graph of the HPLC profile of the sample after renaturation.
FIG. 3 is a graph of the HPLC profile of the sample after renaturation.
FIG. 4 shows an electrophoretogram of a renaturated solution sample after renaturation; lane 1: the molecular weight of the standard protein bands is 100, 30, 25, 20, 15, 10, 5 and 3.4KD from top to bottom; lane 2: boc-human insulin fusion protein inclusion body denaturation lysate; lane 3: detecting reducibility of a renaturation solution of the Boc-human insulin fusion protein; lane 4: non-reducing detection of Boc-human insulin fusion protein renaturation solution;
FIG. 5 is a chart of the HPLC profile of the sample after renaturation.
FIG. 6 is a chart of the HPLC profile of the sample after renaturation.
Detailed Description
The inventors have conducted extensive and intensive studies to obtain, for the first time, a novel Boc-human insulin fusion protein suitable for expression. The fusion protein has the advantages of high folding accuracy, high expression rate and high yield, can be folded at a high concentration in commercial significance, does not need to carry out protein purification on recombinant human insulin fusion protein, directly carries out protein renaturation, and finally obtains 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 renaturation of the recombinant human insulin inclusion body denatured liquid by the renaturation method and the renaturation liquid. On this basis, the inventors completed the present invention.
Terminology
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, when used in reference to a specifically recited value, the term "about" means that the value can vary no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).
As used herein, the term "Tris" is used interchangeably with "Tris (hydroxymethyl) aminomethane".
As used herein, the term "EDTA" is used interchangeably with "ethylenediamine tetraacetic acid".
Unless otherwise indicated, the amino acid sequences of the species Boc-human insulin fusion proteins of the invention are numbered from the N-terminus to the C-terminus.
As used herein, the term "BOC-lysine" is used interchangeably with "t-butoxycarbonyl-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 proteins having the amino acid sequence shown in SEQ ID No.:1 and lysine at position 83 is N epsilon- (t-butoxycarbonyl) -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 obtain the Boc-human insulin with biological activity after enzyme digestion.
As used herein, the term "fusion protein" also includes variants having the above-described activities. These variants include (but are not limited to): deletions, insertions and/or substitutions of 1-3 (typically 1-2, more preferably 1) amino acids, and additions or deletions of one or several (typically within 3, preferably within 2, more preferably within 1) amino acids at the C-terminus and/or N-terminus. For example, in the art, substitution with amino acids of similar or similar properties does not generally alter the function of the protein. As another example, the addition or deletion of one or more amino acids at the C-terminus and/or N-terminus generally does not alter the structure or function of the protein. Furthermore, the term also includes polypeptides of the invention in monomeric and multimeric form. The term also includes linear as well as non-linear polypeptides (e.g., cyclic peptides).
The invention also includes active fragments, derivatives and analogues of the fusion proteins. As used herein, the terms "fragment," "derivative," and "analog" refer to polypeptides that substantially retain the function or activity of the fusion proteins of the invention. The polypeptide fragment, derivative or analogue of the present invention may be (i) a polypeptide having one or several conserved or non-conserved amino acid residues, preferably conserved amino acid residues, substituted or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide formed by fusion of a polypeptide with another compound such as a compound which extends the half-life of the polypeptide, for example polyethylene glycol, or (iv) a polypeptide formed by fusion of an additional amino acid sequence to the polypeptide sequence (fusion protein formed by fusion with a tag sequence such as a leader sequence, a secretory sequence or 6 His). Such fragments, derivatives and analogs are within the purview of one skilled in the art and would be well known in light of the teachings herein.
A preferred class of reactive derivatives refers to polypeptides in which up to 3, preferably up to 2, more preferably up to 1 amino acid is replaced by an amino acid of similar or similar nature, as compared to the amino acid sequence of the invention. These conservatively variant polypeptides are preferably generated 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 differences in amino acid sequence, by differences in modified forms that do not affect the sequence, or by both. Analogs also include analogs having residues other than the natural L-amino acid (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, the fusion proteins of the invention may also be modified. Modified (typically without altering the primary structure) forms include: chemically derivatized forms of polypeptides such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications during synthesis and processing of the polypeptide or during further processing steps. Such modification may be accomplished by exposing the polypeptide to an enzyme that performs glycosylation (e.g., 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 improve their proteolytic resistance or to optimize solubility.
The term "polynucleotide encoding a fusion protein of the invention" may include polynucleotides encoding a fusion protein of the invention, as well as polynucleotides further comprising additional 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 invention. Such nucleotide variants include substitution variants, deletion variants and insertion variants. As 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 it encodes.
Renaturation liquid
The invention provides renaturation solution which is used for renaturation of Boc-human insulin fusion protein denaturation solution after inclusion body cleavage and denaturation of Boc-human insulin fusion protein, and high-yield correctly folded Boc-human insulin fusion protein is obtained. On this basis, the inventors completed the present invention.
In a preferred embodiment of the present invention, the renaturation solution comprises (but is not limited to) Tris, na 2CO3, glycine, naCl, EDTA and cystine.
Specifically, the renaturation solution is as described in the first aspect of the present invention.
Renaturation method
The renaturation solution is used for renaturation treatment of the Boc-human insulin fusion protein.
Typically, the present invention provides a method for renaturation of a Boc-human insulin fusion protein, said method comprising the steps of:
(1) Cracking inclusion bodies containing the Boc-human insulin fusion protein to obtain a cracking mixed solution, and adding a denaturation solution into the cracking mixed solution to obtain a Boc-human insulin fusion protein denaturation solution;
(2) And mixing the modified solution of the Boc-human insulin fusion protein with renaturation solution, and reacting to obtain the renaturated Boc-human insulin fusion protein.
In a preferred embodiment of the invention, the inclusion bodies comprise inclusion bodies of recombinant bacteria expressing the Boc-human insulin fusion protein.
In another preferred embodiment, the recombinant bacterium is E.coli.
In another preferred embodiment, the inclusion bodies are prepared by a method comprising the steps of:
(i) Providing a recombinant bacterium expressing a Boc-human insulin fusion protein;
(ii) And separating inclusion bodies containing the Boc-human insulin fusion protein from the recombinant bacteria.
The main advantages of the invention include
1. The invention unexpectedly develops a renaturation solution and a renaturation method of Boc-human insulin fusion protein, which can obviously improve renaturation efficiency.
2. The recombinant human insulin fusion protein with biological activity is finally obtained by directly carrying out protein renaturation without carrying out 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 increase the solubility of the fusion protein and reduce the intermolecular interaction of the fusion protein, thereby enabling the fusion protein to fold at a commercially significant high concentration.
5. Cyanogen bromide cleavage and oxidative sulfite cleavage and related purification steps are not required in the preparation of the target peptide.
6. The preparation of the target peptide does not require the use of high concentrations of thiols or hydrophobic adsorption resins.
7. The peptides of interest are 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 specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods, in which specific conditions are not noted in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated.
Example 1
1. Construction of recombinant human insulin expression vectors methods of construction are referred to in the art, and in particular may be referred to the description of the examples in patent application number 201910210102.9. The DNA fragment of the fusion protein FP-TEV-R-MiniINS was cloned into the expression vector plasmid pBAD/His A (purchased from NTCC company, kanamycin resistance) at the NcoI-XhoI site downstream of the araBAD promoter, resulting in plasmid pBAD-FP-TEV-R-MiniINS. The plasmid map is shown in FIG. 1.
The pylRs DNA sequence was then cloned into the expression vector plasmid pEvol-pBpF (from NTCC, chloramphenicol resistant) at the site SpeI-SalI downstream of the araBAD promoter, and the DNA sequence of the tRNA of lysyl-tRNA synthetase (pylTcua) was inserted downstream of the proK promoter by PCR. 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 the recombinant escherichia coli strain expressing the Boc-human insulin fusion protein is obtained by 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 rd lysine is N epsilon- (tert-butoxycarbonyl) -lysine.
2. Preparing a seed liquid culture medium, inoculating, performing two-stage culture to obtain a second-stage seed liquid, culturing for 20h, wherein the OD600 reaches about 180, fermenting to obtain about 3L of fermentation liquid, and centrifuging to obtain about 130g/L of wet thalli. After the fermentation liquor is centrifugated, adding a crushing buffer solution, using a high-pressure homogenizer to perform bacteria breaking twice, adding tween 80 with a certain concentration, EDTA-2Na and the like for washing after centrifugation, washing once, and centrifugally collecting sediment to obtain the inclusion body. Approximately 43g of wet weight inclusion bodies per liter of fermentation broth are finally obtained.
3. To refold the fusion protein, the inclusion bodies are dissolved in 8mol/L urea solution having a pH of 9.0-11.0 and containing 10-30 mmol/L mercaptoethanol, so that the concentration of total protein after dissolution is 10-30 mg/mL. The sample is diluted 5 to 10 times and is folded for 16 to 30 hours under the conditions of 4 to 8 ℃ and pH value of 10.5 to 11.8. At room temperature, the pH is maintained at 8.0-9.5, and the fusion protein is digested with trypsin and carboxypeptidase B for 10-20 hours. The reversed phase HPLC analysis result shows that the total yield of renaturation and enzymolysis steps is higher than 75%. Insulin analogues obtained after enzymatic cleavage of trypsin with carboxypeptidase B are designated Boc-human insulin. The sample is clarified by membrane filtration, and the purity of the Boc-human insulin, SDS-polyacrylamide gel electrophoresis, which is initially purified by anion exchange chromatography reaches 90%. The final yield of Boc-human insulin per 1 liter of fermentation broth was about 2.1g. And MALDI-TOF mass spectrometry analysis is carried out on the obtained Boc-human insulin, and the molecular weight of the Boc-human insulin is detected to be consistent with the theoretical molecular weight 5907.7 Da. And eluting and collecting a sample through ion exchange chromatography, adding hydrochloric acid for Boc-human insulin deprotection reaction, adding sodium hydroxide solution for controlling the pH to be 2.0-3.2 to terminate the reaction, and performing two-step high-pressure reverse phase chromatography to obtain the purified recombinant human insulin with the yield higher than 75%, wherein the yield of the recombinant human insulin is about 1100mg per 1 liter of fermentation liquor.
Example 2
In this example, different renaturation methods were examined to renaturate the inclusion bodies of the Boc-modified recombinant human insulin fusion protein of example 1, as follows:
At room temperature, dissolving 20 g of Boc-human insulin fusion protein inclusion body in 500mL of inclusion body dissolving solution containing 8mol/L urea and pH 9.0, and stirring for 0.5-3h at room temperature to ensure that the total protein concentration is about 10g/L; and adding 20mmol/L beta-mercaptoethanol, and adjusting the pH value to 9.0 to obtain the Boc-human insulin fusion protein denatured solution.
The Boc-human insulin fusion protein denatured solution was added to 5000mL of renaturation solution containing 50mmol/L Tris, 10mmol/L Na 2CO3, 50mmol/L glycine, 100mmol/L NaCl,0.5mmol/L ethylenediamine tetraacetic acid (EDTA), 0.5mmol/L L-cystine, and pH was adjusted to 10.0. Air was continuously introduced into the renaturation solution at a rate of 20cm 3/H, and the reaction was stopped after stirring at 4℃for 16 hours.
The renaturation solution is taken and analyzed by HPLC, the HPLC spectrum of the sample after renaturation is shown in figure 2, the retention time of the HPLC spectrum of the correctly folded and renatured Boc-human insulin fusion protein is 5.733min, the quantity of the correctly folded and renatured Boc-human insulin fusion protein actually obtained after renatured is calculated according to the peak area normalization method of the Boc-human insulin fusion protein is 2.43g, and the renatured yield of the Boc-human insulin fusion protein is calculated to be about 81 percent.
Example 3
In this example, different renaturation methods were examined to renaturate the inclusion bodies of the Boc-modified recombinant human insulin fusion protein of example 1, as follows:
At room temperature, dissolving 20g of Boc-human insulin fusion protein inclusion body in 350mL of inclusion body dissolving solution containing 8mol/L urea and having pH of 9.0, and stirring at room temperature for 0.5-3h to ensure that the total protein concentration is about 20g/L; and adding beta-mercaptoethanol with the final concentration of 30mmol/L, and regulating the pH value to 10.0 to obtain the Boc-human insulin fusion protein denatured solution.
The Boc-human insulin fusion protein denatured solution was added to 1750mL of renaturation solution containing 50mmol/L Tris, 10mmol/L Na 2CO3, 50mmol/L glycine, 100mmol/L NaCl,0.5mmol/L EDTA,0.5mmol/L L-cystine at a final concentration, and pH was adjusted to 10.5. Air was continuously introduced into the renaturation solution at a rate of 20cm 3/H, and the reaction was stopped after stirring at 4℃for 16 hours.
And taking renaturation liquid, analyzing and detecting by HPLC, wherein the HPLC diagram of the renaturation liquid sample after renaturation is shown in figure 3, and the SDS-PAGE electrophoresis diagram is shown in figure 4. As can be seen from FIGS. 3 to 4, the retention time of the HPLC profile of the correctly folded and renatured Boc-human insulin fusion protein was 5.749min, the amount of the correctly folded and renatured Boc-human insulin fusion protein actually obtained after the renaturation was calculated according to the peak area normalization method of the Boc-human insulin fusion protein was 2.55g, and the renaturation yield of the Boc-human insulin fusion protein was calculated to be about 85%.
The insulin fusion protein with the retention time of 5.749min and the peak 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 insulin fusion protein is detected to be consistent with the theoretical molecular weight 5907.7 Da.
Example 4
In this example, different renaturation methods were examined to renaturate the inclusion bodies of the Boc-modified recombinant human insulin fusion protein of example 1, as follows:
At room temperature, dissolving 20g of Boc-human insulin fusion protein inclusion body in 350mL of inclusion body dissolving solution containing 8mol/L urea and having pH of 9.0, and stirring at room temperature for 0.5-3h to ensure that the total protein concentration is about 10g/L; and adding 20mmol/L beta-mercaptoethanol to regulate the pH value to 10.0 to obtain the Boc-human insulin fusion protein denatured solution.
The Boc-human insulin fusion protein denatured solution was added to 5000mL of renaturation solution containing 50mmol/L Tris, 10mmol/L Na 2CO3, 50mmol/L glycine, 100mmol/L NaCl,0.5mmol/L EDTA,2.0mmol/L L-cystine at a final concentration, and pH was adjusted to 10.5. Air was continuously introduced into the renaturation solution at a rate of 20cm 3/H, and the reaction was stopped after stirring at 4℃for 16 hours.
HPLC analysis and detection are carried out on renaturation liquid, the HPLC spectrum of a sample after renaturation is shown in figure 5, the retention time of the HPLC spectrum of the correctly folded and renatured Boc-human insulin fusion protein is 5.802min, the quantity of the correctly folded and renatured Boc-human insulin fusion protein actually obtained after renatured is calculated according to the peak area normalization method of the Boc-human insulin fusion protein is 2.34g, and the renatured yield of the Boc-human insulin fusion protein is calculated to be about 78%.
Example 5
In this example, different renaturation methods were examined to renaturate the inclusion bodies of the Boc-modified recombinant human insulin fusion protein of example 1, as follows:
At room temperature, dissolving 20g of Boc-human insulin fusion protein inclusion body in 500mL of inclusion body dissolving solution containing 8mol/L urea and pH 9.0, and stirring for 0.5-3h at room temperature to ensure that the total protein concentration is about 10g/L; and adding 20mmol/L beta-mercaptoethanol to regulate the pH value to 10.0 to obtain the Boc-human insulin fusion protein denatured solution.
The Boc-human insulin fusion protein denatured solution was added to 5000mL of renaturation solution containing 50mmol/L Tris, 10mmol/L Na 2CO3, 50mmol/L glycine, 100mmol/L NaCl,0.5mmol/L EDTA,0.5mmol/L L-cystine at a final concentration, and pH was adjusted to 10.0. The reaction was stopped after stirring at 4℃for 16 hours without introducing air.
The renaturation solution is taken and analyzed by HPLC, the HPLC spectrum of the sample after renaturation is shown in figure 6, the retention time of the HPLC spectrum of the correctly folded and renatured Boc-human insulin fusion protein is 5.810min, the quantity of the correctly folded and renatured Boc-human insulin fusion protein actually obtained after renatured is calculated according to the peak area normalization method of the Boc-human insulin fusion protein is 2.16g, and the renatured yield of the Boc-human insulin fusion protein is calculated to be about 72 percent.
All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.
Sequence listing
<110> Spread of biotechnology Co., ltd
<120> An efficient 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 (31)

1. A method for renaturation of a Boc-human insulin fusion protein, said method comprising the steps of:
(1) Cracking inclusion bodies containing the Boc-human insulin fusion protein to obtain a cracking mixed solution, adding a denaturation solution into the cracking mixed solution, and regulating the pH value to 8.0-11.0 to obtain a Boc-human insulin fusion protein denaturation solution;
Wherein the amino acid sequence of the Boc-human insulin fusion protein is shown as SEQ ID NO. 1, and lysine at 83 th position is N epsilon-t-butoxycarbonyl-lysine;
in the step (1), the denaturing solution comprises a denaturant, wherein the denaturant is beta-mercaptoethanol, and the concentration of the beta-mercaptoethanol is 5-50mmol/L;
(2) Mixing the modified solution of the Boc-human insulin fusion protein with renaturation solution and then reacting to obtain renaturated Boc-human insulin fusion protein;
wherein, the renaturation solution consists of the following components: 20-80mmol/L Tris, 3-20mmol/LNa 2CO3, 20-80mmol/L glycine, 20-250mmol/L NaCl, 0.1-1mmol/L EDTA and 0.1-5mmol/L cystine, water as solvent and pH 9.0-12;
and in the step (2), air is introduced into the reaction system at a rate of 2-100cm 3/H during the reaction.
2. The method of claim 1, wherein the inclusion bodies comprise inclusion bodies of recombinant bacteria expressing a Boc-human insulin fusion protein.
3. The method of claim 1, wherein in step (1), the lysing is performed by adding a lysing solution;
the lysate comprises one or more features selected from the group consisting of:
(1) The pyrolysis liquid comprises 6-10mol/L urea;
(2) The pH value of the lysate is 8.0-10.0;
(3) The weight-volume ratio (g/ml) of the inclusion body to the lysate is 5-40:200-800.
4. The method of claim 1, wherein in step (1), the lysing is performed by adding a lysing solution having a pH of 8.5 to 9.5.
5. The method of claim 1, wherein in step (1), the total protein concentration in the lysis mixture is 10-30g/L.
6. The method of claim 5, wherein in step (1), the total protein concentration in the lysis mixture is 15-25g/L.
7. The method of claim 1, wherein in step (1), the pH is adjusted to 9 to 11 after the denaturing solution is added to the lysis mixture.
8. The method of claim 7, wherein in step (1), the pH is adjusted to 9.5 to 10.5 after the denaturing solution is added to the lysis mixture.
9. The method of claim 1, wherein the volume ratio of renaturation solution to Boc-human insulin fusion protein denaturing solution is 1-10:1.
10. The method of claim 9, wherein the volume ratio of renaturation solution to Boc-human insulin fusion protein denaturation solution is 2-8:1.
11. The method of claim 9, wherein the volume ratio of renaturation solution to Boc-human insulin fusion protein denaturation solution is 3-7:1.
12. The method of claim 1, wherein the concentration of Tris is 30-70mmol/L.
13. The method of claim 12, wherein the concentration of Tris is 40-60mmol/L.
14. The method of claim 13, wherein the concentration of Tris is 45-55mmol/L.
15. The method of claim 1, wherein the renaturation solution has one or more characteristics selected from the group consisting of:
(1) The concentration of Na 2CO3 is 5-15mmol/L;
(2) The concentration of glycine is 30-70mmol/L;
(3) The concentration of NaCl is 50-150mmol/L;
(4) The concentration of EDTA is 0.3-0.7mmol/L;
(5) The concentration of cystine is 0.1-2mmol/L.
16. The method of claim 15, wherein the renaturation solution has one or more characteristics selected from the group consisting of:
(1) The concentration of Na 2CO3 is 8-12mmol/L;
(2) The concentration of glycine is 40-60mmol/L;
(3) The concentration of NaCl is 80-120mmol/L;
(4) The concentration of cystine is 0.1-2mmol/L.
17. The method of claim 16, wherein the renaturation solution has one or more characteristics selected from the group consisting of:
(1) The concentration of the glycine is 45-55mmol/L;
(2) The concentration of NaCl is 90-110mmol/L;
(3) The concentration of cystine is 0.1-1mmol/L.
18. The method of claim 17, wherein the renaturation solution has one or more characteristics selected from the group consisting of:
(1) The concentration of NaCl is 95-105mmol/L;
(2) The concentration of cystine is 0.3-0.7mmol/L.
19. The method of claim 1, wherein the renaturation solution has a pH of 10 to 11.
20. The method of claim 19, wherein the pH of the renaturation solution is between 10.2 and 10.8.
21. The method of claim 20, wherein the pH of the renaturation solution is between 10.3 and 10.7.
22. The method of claim 1, wherein in step (2), the reaction time is 10 to 30 hours; and/or in the step (2), the temperature of the reaction is 0-20 ℃.
23. The method of claim 22, wherein in step (2), the reaction time is 10 to 25 hours.
24. The method of claim 23, wherein in step (2), the reaction time is 12 to 18 hours.
25. The method of claim 22, wherein in step (2), the temperature of the reaction is 3-15 ℃.
26. The method of claim 25, wherein the temperature of the reaction is 2-6 ℃.
27. The method according to claim 1, wherein in the step (2), air is introduced into the reaction system at a rate of 5 to 50cm 3/H during the reaction.
28. The method according to claim 27, wherein in the step (2), air is introduced into the reaction system at a rate of 10 to 40cm 3/H during the reaction.
29. The method according to claim 28, wherein in the step (2), air is introduced into the reaction system at a rate of 10 to 30cm 3/H during the reaction.
30. The Boc-human insulin fusion protein is characterized by having an amino acid sequence shown as SEQ ID NO. 1 and lysine at 83 th position is N epsilon- (tert-butoxycarbonyl) -lysine.
31. An isolated polynucleotide encoding a Boc-human insulin fusion protein according to claim 30.
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