CN116589556B - Preparation method of citrullinated vimentin - Google Patents
Preparation method of citrullinated vimentin Download PDFInfo
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- CN116589556B CN116589556B CN202310850536.1A CN202310850536A CN116589556B CN 116589556 B CN116589556 B CN 116589556B CN 202310850536 A CN202310850536 A CN 202310850536A CN 116589556 B CN116589556 B CN 116589556B
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- 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
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4702—Regulators; Modulating activity
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
- C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The application provides a preparation method of citrullinated vimentin. The preparation method of the application comprises the following steps: (1) expressing recombinant vimentin in a prokaryotic cell; (2) Lysing prokaryotic cells over-expressing recombinant vimentin, and purifying by affinity chromatography to obtain recombinant vimentin solution; (3) And (3) passing the recombinant vimentin solution through a composite chromatographic column, then cleaning the composite chromatographic column by using renaturation liquid, and then dissociating from the composite chromatographic column to obtain citrullinated vimentin, wherein in the step (3), the chromatographic packing in the composite chromatographic column is a mixture of the chromatographic packing coupled with protein arginine deiminase and weak anion exchange chromatographic packing. The method can simplify the preparation flow of the citrullinated vimentin, shorten the preparation time and improve the activity and the yield of the citrullinated vimentin, thereby obviously improving the preparation efficiency of the citrullinated vimentin.
Description
Technical Field
The application belongs to the technical field of protein preparation, and particularly relates to a preparation method of citrullinated vimentin.
Background
Vimentin is a structural protein involved in biological processes such as cell morphology maintenance, signal transduction and cell motility. Citrullination is a post-translational modification of proteins that is involved in the regulation of the immune system and in the regulation of autoimmune responses. Citrullinated vimentin may be associated with the occurrence and progression of a variety of autoimmune diseases, such as rheumatoid arthritis, systemic lupus erythematosus, and sjogren's syndrome, among others.
Studies and applications of citrullinated vimentin are related to a number of fields, mainly including autoimmune disease research, biomarker development, and therapeutic strategy research. Autoimmune diseases research is to develop the association between citrullinated vimentin and autoimmune diseases, and to understand the pathogenesis of these diseases, reveal the roles of citrullinated vimentin in autoimmune reaction and inflammatory process, and provide theoretical basis for diagnosis and treatment of related diseases. Biomarker development refers to that citrullinated vimentin possibly becomes a biomarker of autoimmune diseases, and clinical applications such as early diagnosis, disease typing, disease monitoring, prognosis evaluation and the like are realized by detecting the existence and the expression level of the citrullinated vimentin. The treatment strategy research refers to that citrullinated vimentin possibly becomes a new target point for treating autoimmune diseases, and researchers can utilize a citrullinated vimentin model to carry out drug screening and search a treatment method with potential curative effects on functional recovery or pathological repair of the citrullinated vimentin. In conclusion, the citrullinated vimentin has important background significance in the research and clinical application of autoimmune diseases, and the deep research of the citrullinated vimentin is helpful to promote the diagnosis, treatment and prevention of autoimmune diseases.
At present, two main preparation methods of citrullinated vimentin exist. One is the direct artificial synthesis of citrullinated vimentin, the other is the incubation of recombinant vimentin after mixing with the protein arginine deiminase, followed by multiprocessing to remove the hybrid protein. Both methods have the disadvantage that the artificial synthesis is liable to affect the function, stability and interaction with other molecules, nonspecific and excessive citrullination may lead to loss of protein function or undesired changes in protein structure, which may negatively affect subsequent applications and experiments, and different synthesis conditions and reaction times may lead to different degrees of citrullination, which makes it difficult to compare results between different experiments or laboratories, and thus leads to poor detection accuracy; the mixing incubation method requires more additional steps for purification after enzyme catalysis, the yield and purity of the citrullinated vimentin are difficult to be simultaneously considered, the preparation cost is high, and the protein stability can be influenced by elongating the whole expression purification process and time due to the fact that the citrullinated vimentin is unstable, and further the subsequent application and experiment of the protein can be influenced.
Therefore, there is a need to develop more efficient and high quality methods for preparing citrullinated vimentin, providing a favorable support for the research and clinical application of citrullinated vimentin.
Disclosure of Invention
The application aims to provide a preparation method of citrullinated vimentin, which can simultaneously finish the renaturation, citrullination and purification after citrullination of the vimentin through one-step composite chromatography operation, thereby greatly improving the preparation efficiency of the citrullinated vimentin.
In order to solve the technical problems, the application adopts the following technical scheme:
a method for preparing citrullinated vimentin, comprising the steps of:
(1) Expressing recombinant vimentin in the prokaryotic cell;
(2) Lysing prokaryotic cells over-expressing recombinant vimentin, and purifying by affinity chromatography to obtain recombinant vimentin solution;
(3) The recombinant vimentin solution is passed through a composite chromatographic column, then the composite chromatographic column is washed by using renaturation liquid, and then citrullinated vimentin is obtained by dissociation from the composite chromatographic column,
in the step (3), the chromatographic packing in the composite chromatographic column is a mixture of the chromatographic packing coupled with protein arginine deiminase and weak anion exchange chromatographic packing; the formula of the renaturation solution is as follows: 10-50 mM Tris (hydroxymethyl) aminomethane (Tris), 1-10 mM calcium chloride (CaCl) 2 ) 0.5-2 mM ethylenediamine tetraacetic acid (EDTA), 1-10 mM methyl ammonium chloride, and the balance water.
Further preferably, the renaturation solution comprises the following formula: 15-30 mM of tris (hydroxymethyl) aminomethane, 4-8 mM of calcium chloride, 0.5-1.5 mM of ethylenediamine tetraacetic acid, 4-8 mM of methyl ammonium chloride and the balance of water.
Further preferably, the pH value of the renaturation solution is 7-7.5.
Preferably, in the step (3), the volume ratio of the chromatographic packing coupled with the protein arginine deiminase to the weak anion exchange chromatographic packing is 1: (2-5), for example, 1:2. 1:2.5, 1:3. 1:3.5, 1:4. 1:4.5, 1:5.
preferably, the chromatographic packing coupled with the protein arginine deiminase is agarose particles coupled with the protein arginine deiminase, and the weak anion exchange chromatographic packing is ANX weak anion exchange agarose particles.
According to some embodiments, the method for preparing the protein arginine deiminase coupled agarose microparticles comprises: the agarose microparticles were activated using 1-ethyl- (3-dimethylaminopropyl) carbodiimide and the activated agarose microparticles were incubated with the protein arginine deiminase at room temperature. Preferably, the agarose microparticles are preactivated with NHS. The agarose particles preferably have an average particle diameter of 80 to 100 μm.
Preferably, in the step (3), the composite chromatographic column is cleaned at a speed of 0.8-3 mL/min by using a renaturation solution with a volume which is 5-10 times that of the column.
Preferably, in the step (3), the dissociation adopts a renaturation solution and a renaturation solution added with 0.5-2M sodium chloride to carry out dissociation according to a linear gradient, wherein the renaturation solution is from 100% to 0%, and the renaturation solution added with 0.5-2M sodium chloride is from 0% to 100%.
Preferably, in step (1), the recombinant vimentin is histidine-tagged; in step (2), the affinity chromatography purification is performed using a nickel column.
Preferably, the prokaryotic cell is E.coli.
Preferably, in the step (1), pET-14b plasmid connected with the vimentin gene fragment is transfected into an escherichia coli strain, escherichia coli containing the vimentin expression plasmid is cultured, and isopropyl-beta-D-thiogalactoside is added into bacterial liquid to induce the vimentin to be overexpressed.
Preferably, in the step (2), the prokaryotic cells over-expressing the recombinant vimentin are lysed by lysozyme, inclusion bodies are separated, the inclusion bodies are dissolved by 5-10M urea solution, the mixture is centrifuged, and the supernatant is taken to pass through a nickel column and dissociated to obtain the recombinant vimentin solution.
The application also provides citrullinated vimentin prepared by the preparation method.
Compared with the prior art, the application has the following advantages:
the preparation method can simultaneously finish the renaturation, citrullination and purification after citrullination of the recombinant vimentin through one-step composite chromatography operation, thereby greatly improving the preparation efficiency of the citrullinated vimentin; by selecting a proper renaturation solution, the activity of citrullinated vimentin is improved.
Drawings
FIG. 1 is an SDS-PAGE electrophoresis of citrullinated vimentin of example 1;
FIG. 2 is an immunochromatographic representation of citrullinated vimentin and natural vimentin of example 1;
FIG. 3 is a graph comparing the activities of citrullinated vimentin and natural vimentin of example 1;
FIG. 4 is a graph comparing the activity of citrullinated vimentin of example 1 and comparative example 2;
FIG. 5 is a graph comparing the activity of citrullinated vimentin of example 1 and comparative example 3.
Detailed Description
After the vimentin is over-expressed, the vimentin is enriched in the inclusion body, and a plurality of steps such as dissolution, renaturation, citrullination, separation and purification and the like are needed, so that the whole process is complex, the loss of the vimentin is large, and the yield is low. Since citrullinated vimentin is a protein which is easily polymerized and depolymerized, its stability is poor, and the longer the operation time, the lower the activity of citrullinated vimentin. In addition, the selection of renaturation solution is also critical to the activity of citrullinated vimentin. According to the application, through a composite chromatography mode, the dissolved inclusion body is filled with a chromatography filler coupled with protein arginine deiminase and a weak anion exchange chromatography filler, and is eluted by a renaturation solution with a special formula, and the renaturation, citrullination and purified vimentin are obtained through dissociation, so that the renaturation, citrullination and separation and purification steps are successfully integrated into one-step composite chromatography, the operation is simplified, the operation duration is shortened, the yield and activity of the citrullinated vimentin are improved, and the production effect of the citrullinated vimentin is further remarkably improved.
Specifically, the preparation method of the application comprises the following steps:
step 1: cloning genes.
The protein sequence of the vimentin is optimized according to the priority level of the expression of the escherichia coli, a His tag is added, then the gene corresponding to the vimentin is digested by restriction enzymes NdeI and BamHI, and then the gene is cloned into an expression plasmid pET-14b by using a ligase, and the pET-14b plasmid connected with the vimentin gene fragment is transfected into an escherichia coli strain BL21 DE 3.
Step 2: coli comprising the vimentin expression plasmid was cultured.
Inoculating positive colonies into LB culture solution (containing 50 mug/mL ampicillin) for culture, growing bacteria until the OD600 value of the culture solution is 0.8-1.0, adding isopropyl-beta-D-thiogalactoside into the bacterial solution to induce vimentin to express, and centrifuging to collect bacterial blocks containing expressed vimentin.
Step 3: cell disruption and lysis.
Firstly, using lysozyme and an ultrasonic crushing mode to crack fungus blocks, then using urea solution to dissolve inclusion bodies, uniformly mixing the dissolved solution, and centrifuging to obtain supernatant.
Step 4: and purifying the protein by using a nickel ion affinity chromatography column.
The supernatant from step 3 is passed through a nickel ion affinity column, preferably with a solution of 20mM Tris,8M urea, 5mM methyl ammonium chloride to wash 5 column volumes, followed by dissociation of vimentin with a solution of 20mM Tris,8M urea, 5mM methyl ammonium chloride, 250mM imidazole.
Step 5: protein arginine deiminase was coupled to agarose microparticles.
NHS pre-activated agarose microparticles with a microparticle size of 90 μm were selected and washed with phosphate buffer to remove potential contaminants and residues. The washing step may be repeated 2-3 times. The agarose particles were activated with 1-ethyl- (3-dimethylaminopropyl) carbodiimide at a final concentration of 5mM, and after incubation of protein arginine deiminase and activated agarose particles at a mass ratio of 1:50 for 1 hour, the coupled agarose particles were washed with phosphate buffer to remove unreacted activator and other impurities. The washing step may be repeated 2-3 times. Thereby obtaining agarose particles coupled with protein arginine deiminase.
Step 6: vimentin renaturation and citrullination.
Agarose microparticles conjugated with protein arginine deiminase were mixed with ANX weak anion exchange agarose microparticles according to 1: (2-5) by volume ratio. Loading the vimentin obtained by dissociation in step 4 onto column, and then subjecting to 5-10 column volumes of renaturation solution (preferable formula: 20mM Tris,5mM CaCl) 2 1mM EDTA,5mM methyl ammonium chloride, balance water). The flow rate of the renaturation solution is not more than 3 mL/min. And after the cleaning is finished, dissociating the solution according to a linear gradient by using a renaturation solution and a renaturation solution containing 1M sodium chloride, wherein the renaturation solution is from 100% to 0%, and the renaturation solution containing 1M sodium chloride is from 0% to 100%, so that the high-purity citrullinated vimentin with finished renaturation is obtained.
The application is further described below with reference to examples. The present application is not limited to the following examples. The implementation conditions adopted in the embodiments can be further adjusted according to different requirements of specific use, and the implementation conditions which are not noted are conventional conditions in the industry. The technical features of the various embodiments of the present application may be combined with each other as long as they do not collide with each other.
In the following examples, unless otherwise specified, the raw materials, reagents and the like used were all conventional commercial products. Agarose microparticle suspensions were purchased from sitz, usa (cytova), cat No.: 17090602 the agarose particles were preactivated with NHS, the average particle size was 90 μm, the solids content was 40mg/mL, and the stock solution was 100% isopropyl alcohol. Protein arginine deiminase was purchased from Sigma Aldrich, cat: SAE0086.ANX weak anion exchange agarose microparticle suspensions were purchased from smithing (cytova), cat No.: 17128701 the average particle size is 90 μm, the solid content is 40mg/mL, and the preservation solution is 20% ethanol. Capto-Q strong anion exchange agarose particle suspensions were purchased from Situo (Cytiva), U.S. under the designation: 17531602 the average particle size is 90 μm and the preservation solution is 20% ethanol. DEAE anion exchange chromatography column was purchased from Situo, U.S.A. (Cytiva), cat: 17515401.HIPREP 16/60 SEPHACRYL S-100HR molecular sieve chromatography column was purchased from Situo Va (Cytiva), cat: 17116501.
example 1
The embodiment provides a preparation method of citrullinated vimentin, which comprises the following specific steps:
step 1: cloning genes.
Querying NCBI protein information base to obtain protein sequence of human source waveform protein with NP 003371 (from amino acid No. 1 to 465):
MSTRSVSSSSYRRMFGGPGTASRPSSSRSYVTTSTRTYSLGSALRPSTSRSLYASSPGGVYATRSSAVRLRSSVPGVRLLQDSVDFSLADAINTEFKNTRTNEKVELQELNDRFANYIDKVRFLEQQNKILLAELEQLKGQGKSRLGDLYEEEMRELRRQVDQLTNDKARVEVERDNLAEDIMRLREKLQEEMLQREEAENTLQSFRQDVDNASLARLDLERKVESLQEEIAFLKKLHEEEIQELQAQIQEQHVQIDVDVSKPDLTAALRDVRQQYESVAAKNLQEAEEWYKSKFADLSEAANRNNDALRQAKQESTEYRRQVQSLTCEVDALKGTNESLERQMREMEENFAVEAANYQDTIGRLQDEIQNMKEEMARHLREYQDLLNVKMALDIEIATYRKLLEGEESRISLPLPNFSSLNLRETNLDSLPLVDTHSKRTLLIKTVETRDGQVINETSQHHDDLE（SEQ ID No.1）
adding 6 histidine tags at the N-terminal of the human source waveform protein, and optimizing the protein sequence of the human source waveform protein according to the priority level of escherichia coli expression, wherein the obtained gene sequence is as follows:
ATGAGCACCCGTAGCGTTTCTAGCTCTTCTTATCGTCGTATGTTTCGTGGCCCAGGTACCGCGAGCCGTCCGTCTAGCTCTCGTAGCTACGTTACCACCTCTACCCGCACCTACAGCCTGGGCAGCGCGCTGCGTCCGAGCACCTCTCGTAGCCTGTACGCAAGCTCTCCGGGCCGTGTTTACGCGACCCGCAGCTCCGCTGTTCGTCTGCGTTCTTCTGTTCCGGGTGTTCGTCTGCTGCAGGACTCGGTTGATTTCTCTCTGGCGGACGCGATCAACACCGAATTCAAAAACACCCGCACTAACGAAAAAGTGGAACTGCAGGAACTGAACGATCGTTTCGCTAACTATATCGATAAAGTGCGTTTCCTGGAACAGCAGAACAAAATTCTTCTGGCTGAACTGGAACAGCTGAAAGGTCAGGGTAAATCCCGTCTGGGCGATCTGTACGAAGAAGAAATGCGTGAACTGCGTCGTCAGGTTGATCAGCTGACCAACGACAAAGCGCGTGTTGAAGTGGAACGTGATAACCTGGCGGAAGATATCATGCGCCTGCGTGAAAAACTGCAGGAAGAAATGCTGCAACGCGAAGAAGCGGAAAACACCCTGCAGTCTTTCCGCCAGGATGTTGATAACGCCTCTCTGGCGCGTCTGGACCTGGAACGCAAAGTTGAAAGCCTGCAGGAAGAAATCGCGTTCCTGAAAAAACTGCACGAAGAAGAAATCCAGGAACTGCAGGCTCAGATCCAGGAACAGCACGTTCAGATTGATGTTGATGTGAGCAAACCGGATCTGACCGCGGCCCTGCGTGATGTGCGTCAGCAGTATGAAAGCGTTGCTGCGAAAAACCTGCAGGAAGCAGAAGAATGGTATAAATCTAAATTCGCAGATCTGTCCGAAGCAGCGAACCGCAACAACGATGCGCTGCGTCAGGCGAAACAGGAAAGCACCGAATACCGTCGCCAGGTACAGTCTCTGACCTGCGAAGTTGATGCGCTGAAAGGCACTAACGAAAGCCTGGAACGTCAGATGCGCGAAATGGAAGAAAACTTCGCAGTTGAAGCAGCGAACTACCAGGATACCATCGGCCGCCTGCAGGATGAAATCCAGAACATGAAAGAAGAAATGGCTCGTCATCTGCGTGAATACCAGGACCTGCTGAACGTCAAAATGGCTCTGGATATTGAAATCGCTACCTATCGCAAACTGCTGGAAGGTGAAGAAAGCCGTATTAGCCTGCCGCTGCCGAACTTCAGCAGCCTGAACCTGCGTGAAACTAACCTGGATAGCCTGCCGCTGGTTGATACCCACTCTAAACGTACCCTGCTGATTAAAACTGTTGAAACTCGTGATGGTCAGGTTATCAACGAAACCTCCCAGCACCACGATGATCTGGAACACCACCACCACCACCACTAA（SEQ ID No.2）
NdeI restriction enzyme cutting site is added to the 5 'end of the nucleic acid sequence, and BamHI restriction enzyme cutting site is added to the 3' end. 1. Mu.g of pET-14b plasmid and vimentin nucleic acid fragment were digested with 5 units of NdeI and BamHI restriction enzymes, respectively, at 37℃for 1 hour. The digested plasmid and vimentin nucleic acid fragments were combined according to 1:3, 10 units of T4 ligase was added, and the mixture was ligated at 25℃for 1 hour and transferred into BL21 DE3 E.coli. Positive colonies were picked on LB plates mixed with 50. Mu.g/mL ampicillin Lin Kangsheng.
Step 2: vimentin expression.
Inoculating positive colonies into LB culture solution (containing 50 mug/mL ampicillin) with the volume of 1L for culture, growing bacteria until the OD600 value of the culture solution is between 0.8 and 1.0, adding isopropyl-beta-D-thiogalactoside with the final concentration of 1mM into the culture solution, continuously carrying out shaking culture for 3 hours, centrifuging the bacterial solution at 8000rpm for 10 minutes, and collecting bacterial block precipitates.
Step 3: cell disruption and lysis.
The bacterial lysate is used for dissolving bacterial block sediment, and the formula of the bacterial lysate is as follows: 50mM Tris,1mM EDTA,300mM NaCl,5wt% glycerol, 100 μg/mL lysozyme, one piece of protease inhibitor per 50mL, and the balance water. Ultrasound was then used to break the bacteria, with the ultrasound being performed in an ice bath, with 10 seconds of suspension every 10 seconds of ultrasound, and 30 minutes of full ultrasound. The sonicated resuspension was centrifuged at 12000rpm for 10 minutes, and the inclusion body pellet was taken. The inclusion body pellet was rewashed 3 times with bacterial lysate, and after completion of the 3 times washing, the inclusion body was dissolved in 10mL of a solution (20 mM Tris,8M urea, 5mM methyl ammonium chloride). After the inclusion bodies were homogenized, they were centrifuged at 15000rpm for 10 minutes, and the supernatant containing vimentin was collected.
Step 4: the vimentin was separated by a nickel ion affinity column.
The supernatant containing vimentin was passed through a nickel ion affinity column (10 mL) equilibrated with an equilibration solution (20 mM tris,8m urea, 5mM methyl ammonium chloride, balance water) and after the supernatant had all flowed, the column was washed 5 column volumes with a washing solution (20 mM tris,8m urea, 5mM methyl ammonium chloride, balance water, ph 7.4). After the completion of the washing, vimentin was dissociated using a dissociation solution (20 mM tris,8m urea, 5mM methyl ammonium chloride, 250mM imidazole, the balance of water, pH 7.4) to total 10 fractions each having a volume of 5mL, the amount and purity of the protein were judged by running SDS-PAGE electrophoresis for all the fractions, and the fractions containing the vimentin of interest were selected and mixed to obtain vimentin for the subsequent step.
Step 5: protein arginine deiminase was coupled to agarose microparticles.
Taking 12.5mL of agarose particle suspension (mixing reversely before use), flushing agarose particles by using 50mL of phosphate buffer PBS, centrifuging for 10 minutes at 8000rpm, discarding supernatant, collecting agarose particles, repeating flushing and centrifuging steps for 3 times, re-suspending the cleaned agarose particles by using the phosphate buffer PBS, adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide with the final concentration of 5mM, incubating for 1 hour at room temperature, mixing reversely after incubation is finished, centrifuging for 10 minutes at 8000rpm, discarding supernatant, collecting agarose particles, re-suspending by using 10mL of phosphate buffer PBS, adding 10mg of protein arginine deiminase, mixing uniformly at room temperature, and incubating for 1 hour at room temperature. Transferring the incubated agarose particles solution to a gravity chromatographic column, adding 30mL of phosphate buffer PBS to wash the agarose particles until the agarose particles completely flow out of the phosphate buffer PBS, and repeatedly washing for 3 times to obtain the agarose particles coupled with the protein arginine deiminase, and re-suspending and storing the agarose particles in 10mL of phosphate buffer PBS.
Step 6: vimentin renaturation and citrullination.
10mL of the protein arginine deiminase-coupled agarose microparticle suspension was mixed with 30mL of the ANX weak anion exchange agarose microparticle suspension (mixed upside down before use), the mixture was packed into a column, and the column was washed with 200mL of 20mM Tris-HCl, pH7.4 buffer solution. The vimentin solution dissociated from the nickel column was passed through a mixing agarose microparticle column with a loading of 25mL, followed by 200mL of renaturation solution (20mM Tris,5mM CaCl 2 1mM EDTA,5mM methyl ammonium chloride, balance water, pH 7.4) was used to wash the column, the washing flow rate was set at 2 mL/min. After the column was washed, the solution A (20mM Tris,5mM CaCl 2 1mM EDTA,5mM methyl ammonium chloride, balance water, pH 7.4) and solution B (20mM Tris,5mM CaCl 2 1mM EDTA,5mM methyl ammonium chloride, 1M sodium chloride, the balance of water, pH 7.4) were mixed in a linear gradient in 100mL so that the solution A was from the first 100% to the final 0%, the solution B was from 0% to 100%, the capacity of each of the collected components was 5mL, a total of 20 components were collected, the amount and purity of the protein were judged by running SDS-PAGE electrophoresis, and the components containing the objective vimentin and having a high vimentin purity were selected and mixed as the final collected citrullinated vimentin. According to the method, 0.7-0.8 mg of citrullinated vimentin can be obtained after renaturation and purification of every 1mg of vimentin, and the yield is more than 70%.
And (3) activity verification:
(1) The citrullinated vimentin thus collected was subjected to SDS-PAGE to obtain a protein having a molecular weight of about 55kDa, the protein purity being 90% or more (FIG. 1).
(2) The activity of citrullinated vimentin was verified by immunoblotting experiments: citrullinated vimentin after completion of SDS-PAGE electrophoresis was transferred onto PVDF membrane, and PVDF membrane was blocked with phosphate buffer PBS containing 1% bovine serum albumin for 1 hour, followed by 1:1000 volume ratio anti-vimentin monoclonal antibody solution diluted in phosphate buffer PBS was incubated with PVDF membrane for 1 hour, followed by 1: the PVDF membrane was incubated with horseradish peroxidase-conjugated goat anti-mouse IgG solution diluted in phosphate buffer PBS for 1 hour in 5000 volume ratio, and washed 3 times with phosphate buffer PBS before each incubation. Finally, 2mL of ECL color development liquid is added on the cleaned PVDF film, the PVDF film is observed in an immunoblotting imager after 1 minute exposure, the waveform protein with the corresponding molecular weight can be clearly seen to be displayed on the film (the citrullinated waveform protein is on the left side in the figure 2 and the natural waveform protein is on the right side in the figure 2), and the result shows that the molecular weight of the citrullinated waveform protein is equivalent to that of the natural waveform protein.
(3) The activity of the expressed citrullinated vimentin was verified using an enzyme-linked immunosorbent assay: the obtained citrullinated vimentin and natural vimentin are respectively coated on the ELISA plate according to the amount of 0.2 mug/hole, and after being coated for 2 hours at 37 ℃, the ELISA plate is cleaned for 2 times by using phosphate buffer PBS. Each well was blocked with 100 μl of phosphate buffer PBS containing 1% bovine serum albumin for 1 hour at room temperature. The elisa plate was washed 5 times with phosphate buffer PBS. Each sample was repeated for 5 wells, wherein 100 μl of 100 μg/mL,10 μg/mL,1 μg/mL,0.1 μg/mL of anti-citrullinated vimentin murine monoclonal antibody was added to 4 wells, and after incubation for 1 hour at room temperature, the elisa plate was washed 5 times with phosphate buffer PBS. Each hole is added with 100 mu L of volume dilution ratio of 1:1000 horseradish peroxidase-conjugated goat anti-mouse IgG, after incubation for 1 hour at room temperature, the ELISA plate was washed 5 times with phosphate buffer PBS. Finally, adding 100 mu L of TMB substrate, incubating for 10 minutes, adding 50 mu L of 2M concentrated sulfuric acid, stopping the reaction, and reading absorbance at 450 nm. The results of the immunoassay are shown in fig. 3, and fig. 3 shows that the citrullinated vimentin prepared in example 1 has higher reactivity with the anti-citrullinated vimentin antibody than the natural vimentin.
Comparative example 1
This comparative example provides a method for preparing citrullinated vimentin, which is substantially the same as example 1, except that steps 5 and 6 are adjusted to: vimentin obtained by dissociation from nickel columnThe solution was passed through a DEAE anion exchange chromatography column at a loading of 25mL followed by 200mL of renaturation solution (20mM Tris,5mM CaCl 2 1mM EDTA,5mM methyl ammonium chloride, balance water, pH 7.4) was used to wash the column, the washing flow rate was set at 2 mL/min. After renaturation was completed, solution A (20mM Tris,5mM CaCl 2 5mM methyl ammonium chloride, balance water, pH 7.4) and solution B (20mM Tris,5mM CaCl 2 5mM methyl ammonium chloride, 1M sodium chloride, the balance water, pH 7.4) was mixed in a linear gradient in 100mL to dissociate renaturated vimentin from solution A from initial 100% to final 0% and solution B from 0% to 100%. Mixing protein arginine deiminase at a ratio of 50 units of enzyme per 1mg protein, incubating at room temperature for 3 hr, adding EGTA with final concentration of 50mM to buffer solution after 3 hr, stopping reaction at pH of 8.0, and dialyzing the citrullinated vimentin mixed solution (dialysate formula: 20mM Tris,5mM CaCl) 2 In 5mM methyl ammonium chloride, 250mM sodium chloride, the remainder water, pH 7.4), the dialyzed solution was subjected to Hiprep 16/60 Sephacryl S-100HR molecular sieve column (pre-packed allyl dextran/bisacrylamide cross-packing) using a dialysate (formulation: 20mM Tris,5mM CaCl 2 5mM methyl ammonium chloride, 250mM sodium chloride, the rest of water, pH 7.4) are dissociated and collected, and the components containing citrullinated vimentin are mixed according to the chromatographic profile and the SDS-PAGE electrophoresis result and collected to obtain the citrullinated vimentin. The citrullinated vimentin obtained by the method has low yield, the content of the vimentin obtained by dissociation from the nickel ion column per 1mg is lower than 0.5mg after renaturation and purification, and the protein yield is lower than 50%.
Comparative example 2
This comparative example provides a method for preparing citrullinated vimentin, which is substantially the same as example 1, except that steps 5 and 6 are adjusted to: the vimentin solution dissociated from the nickel column was dialyzed sequentially in a dialysate containing 6M urea, 4M urea, 2M urea, 0M urea (dialysate formulation: urea, 5mM Tris,1mM DTT,1mM EDTA, balance water, ph 8.4). Each dialysis was performed in 5L of dialysate and dialyzed for 20 hours. The dialyzed wave-shaped eggsThe white solution was applied to a 5mL ANX weak anion exchange agarose column using 25mL 20mM Tris,5mM CaCl 2 The column was rinsed with 5mM methyl ammonium chloride, pH7.4 buffer solution, at a flow rate of 1 mL/min. After the column was washed, the solution A (20mM Tris,5mM CaCl 2 1mM EDTA,5mM methyl ammonium chloride, balance water, pH 7.4) and solution B (20mM Tris,5mM CaCl 2 1mM EDTA,5mM methyl ammonium chloride, 1M sodium chloride, the balance of water, pH 7.4) were mixed in a linear gradient in 100mL so that the solution A was from the first 100% to the final 0%, the solution B was from 0% to 100%, the capacity of each collected component was 1mL, 10 components in total were collected, the amount and purity of the protein were judged by running SDS-PAGE, and the components containing the objective vimentin and having high vimentin purity were selected and mixed as vimentin. Mixing the vimentin purified by ANX ion exchange chromatography column according to the proportion of 50 units of protein arginine deiminase per 1mg of protein, incubating at room temperature for 3 hours, adding EGTA with final concentration of 50mM into buffer solution after 3 hours, stopping reaction at pH of 8.0, and dialyzing the citrullinated vimentin mixed solution (dialysate formula: 20mM Tris,5mM CaCl) 2 In 5mM methyl ammonium chloride, 250mM sodium chloride, the remainder water, pH 7.4), the dialyzed solution was subjected to Hiprep 16/60 Sephacryl S-100HR molecular sieve column (pre-packed allyl dextran/bisacrylamide cross-packing) using a dialysate (formulation: 20mM Tris,5mM CaCl 2 5mM methyl ammonium chloride, 250mM sodium chloride, the rest of water, pH 7.4) are dissociated and collected, and the components containing citrullinated vimentin are mixed according to the chromatographic profile and the SDS-PAGE electrophoresis result and collected to obtain the citrullinated vimentin.
The citrullinated vimentin obtained by the method has long time consumption and poor renaturation effect. The citrullinated vimentin prepared in comparative example 2 and the citrullinated vimentin prepared in example 1 were compared for activity using an enzyme-linked immunosorbent assay (the method of operation is basically the same as example 1, except that the natural vimentin was replaced with the citrullinated vimentin prepared in comparative example 2, and the concentration of the anti-citrullinated vimentin murine monoclonal antibody was set at 10 μg/mL,1 μg/mL and 0.1 μg/mL). The results of the immunoassay are shown in fig. 4, and fig. 4 shows that the reactivity of citrullinated vimentin and antibody prepared in example 1 is significantly higher than that of citrullinated vimentin prepared in comparative example 2 at the same coating concentration.
Comparative example 3
This comparative example provides a method for citrullinating vimentin which is substantially the same as example 1, except that the formulation of the renaturation solution used in step 6 is as follows: 20mM Tris,5mM CaCl 2 1mM EDTA,20mM urea, 1mM EGTA,20mM L-arginine, the balance water, pH7.4.
The citrullinated vimentin prepared in comparative example 3 and the citrullinated vimentin prepared in example 1 were compared for activity using an enzyme-linked immunosorbent assay (the method of operation is basically the same as example 1, except that the natural vimentin was replaced with the citrullinated vimentin prepared in comparative example 3, and the concentration of the anti-citrullinated vimentin murine monoclonal antibody was set at 10 μg/mL,1 μg/mL and 0.1 μg/mL). The results of the immunoassay are shown in fig. 5, and fig. 5 shows that the reactivity of citrullinated vimentin and antibody prepared in example 1 is significantly higher than that of citrullinated vimentin prepared in comparative example 3 at the same coating concentration.
Comparative example 4
This comparative example provides a method for preparing citrullinated vimentin, which is substantially identical to example 1, except that the ANX weak anion-exchange sepharose particles suspension in step 6 is replaced by Capto-Q strong anion-exchange sepharose particles suspension. By adopting the preparation method of the comparative example, the yield of the vimentin obtained by dissociation from the nickel ion column after renaturation and purification is only 0.3-0.4 mg, the protein yield is lower than 40%, and is far lower than that of the example 1.
The present application has been described in detail with the purpose of enabling those skilled in the art to understand the contents of the present application and to implement the same, but not to limit the scope of the present application, and all equivalent changes or modifications made according to the spirit of the present application should be included in the scope of the present application.
Claims (7)
1. A method for preparing citrullinated vimentin, which is characterized by comprising the following steps: the method comprises the following steps:
(1) Inducing escherichia coli to overexpress recombinant vimentin by adopting isopropyl-beta-D-thiogalactoside;
(2) Splitting escherichia coli over-expressing recombinant vimentin, and performing affinity chromatography purification by using a nickel column to obtain a recombinant vimentin solution;
(3) The recombinant vimentin solution passes through a composite chromatographic column, then the composite chromatographic column is cleaned by using renaturation liquid, and then citrullinated vimentin is obtained by dissociation from the composite chromatographic column, in the step (3), chromatographic packing in the composite chromatographic column is a mixture of agarose particles coupled with protein arginine deiminase and ANX weak anion exchange agarose particles; the formula of the renaturation solution is as follows: 10-50 mM of tris (hydroxymethyl) aminomethane, 1-10 mM of calcium chloride, 0.5-2 mM of ethylenediamine tetraacetic acid, 1-10 mM of methyl ammonium chloride and the balance of water.
2. The method of manufacturing according to claim 1, characterized in that: in the step (3), the volume ratio of the protein arginine deiminase coupled agarose particles to the ANX weak anion exchange agarose particles is 1: (2-5).
3. The method of manufacturing according to claim 1, characterized in that: in the step (3), the composite chromatographic column is cleaned at a speed of 0.8-3 mL/min by using a renaturation solution with a volume which is 5-10 times that of the column.
4. The method of manufacturing according to claim 1, characterized in that: in the step (3), the dissociation adopts a renaturation solution and a renaturation solution added with 0.5-2M sodium chloride to carry out dissociation according to a linear gradient.
5. The method of manufacturing according to claim 1, characterized in that: in step (1), the recombinant vimentin is histidine-tagged.
6. The method of manufacturing according to claim 1, characterized in that: in the step (1), pET-14b plasmid connected with the vimentin gene fragment is transfected into an escherichia coli strain, escherichia coli containing the vimentin expression plasmid is cultured, and isopropyl-beta-D-thiogalactoside is added into bacterial liquid to induce the vimentin to be overexpressed.
7. The method of manufacturing according to claim 1, characterized in that: in the step (2), the prokaryotic cells which over-express the recombinant vimentin are cracked by using lysozyme, inclusion bodies are separated, the inclusion bodies are dissolved by using 5-10M urea solution, the mixture is centrifuged, supernatant fluid is taken to pass through a nickel column, and the recombinant vimentin solution is obtained by dissociation.
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