CN114058636A - Method for cloning, expressing and purifying transthyretin gene - Google Patents

Abstract

The invention discloses a method for cloning, expressing and purifying TTR gene, which comprises the steps of obtaining TTR recombinant plasmid by chemical synthesis and double enzyme digestion, inducing protein to express in large quantity, salting out, buffer solution replacement dialysis, anion exchange column and gel filtration chromatography column to obtain TTR protein with purified precision. The protein purification method is based on an AKTA protein purification system, is simple and convenient to operate, can simultaneously realize automatic washing, online monitoring, real-time observation and the like of a sample, can adjust the purification process in time according to the condition of the sample and the subsequent requirement on the purified sample, obtains the TTR protein with high purity, can save precious plasma resources to the maximum extent, and simultaneously lays a foundation for the in vitro research of TTR genes in future.

Description

Method for cloning, expressing and purifying transthyretin gene

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for cloning, prokaryotic expression and protein purification of a transthyretin (TTR) gene.

Technical Field

Transthyretin (TTR), also known as Prealbumin (PA), is a tetrameric protein formed by 4 identical subunits connected by non-covalent bonds, each subunit consisting of 127 amino acids and having a relative molecular mass of 55 kDa. In vivo, TTR is produced primarily by the choroid plexus of the liver and brain, and also is synthesized in small amounts by retinal epithelial cells, primarily in blood and cerebrospinal fluid, at levels of about 3.4-5. mu. mol/L in blood and about 0.04-0.4. mu. mol/L in cerebrospinal fluid. The protein has the main physiological functions of transferring thyroxine and transferring vitamin A by combining with retinol binding protein.

Under normal physiological conditions, TTR functions as a stable tetramer, but when a gene coding for TTR is mutated or grows with age, the protein tetramer structure of TTR is unstable, TTR monomer pathologically aggregates in tissues such as peripheral nervous system, heart, eye, kidney, meninges and the like to form insoluble amyloid deposition, and transthyretin Amyloidosis (ATTR) disease is caused. Research reports that ATTR diseases have the characteristics of low morbidity, progressive exacerbation, various clinical manifestations, great difficulty in clinical diagnosis and differential diagnosis and the like, the survival period of patients after confirmed diagnosis is only 2-12 years generally, and most of patients can die from heart diseases.

Currently, in clinical treatment of ATTR disease, liver transplantation has become the first line of treatment, and not only can the variant TTR be reduced, but also the overall survival rate can be improved. However, liver transplantation has the effects of donor shortage, operation risk, acute rejection, long-term use of immunosuppressive agents, patient condition limitation and the like. In recent years, given the increasing maturity of protein expression and purification techniques and protein stability research techniques, more and more research teams have focused therapeutic strategies for ATTR disease on inhibiting the dissociation of TTR tetramers, i.e. slowing the rate of amyloid formation. Therefore, the efficient purification scheme of the TTR protein has significant practical significance and application value for the research and treatment of diseases.

At present, the reported TTR protein extraction and purification methods (EP0711786a2, US5310878a1) disclose a method for purifying human plasma prealbumin, which requires 11 extraction steps including ion exchange, immunodiffusion, affinity chromatography, size exclusion chromatography, and multiple buffer replacement and concentration steps under specific conditions. In addition, CN103833843A discloses a method for extracting and purifying prealbumin PA from normal human plasma, which requires solution extraction, buffer solution replacement dialysis, and purification of three kinds of chromatographic columns in AKTA system. The methods directly extract and purify TTR protein from human plasma, and have the disadvantages of complicated steps and complex operation, thus being difficult to meet the requirements of industrial production or clinical research.

Disclosure of Invention

In view of the limitation of the existing TTR protein purification, the invention provides a brand new TTR purification method, which comprises the steps of obtaining TTR recombinant plasmids by chemical synthesis and double enzyme digestion, inducing protein to express in large quantity, primarily purifying TTR protein by salting out, buffer solution replacement dialysis, anion exchange column and other steps, and finally obtaining highly purified TTR protein by matching with a gel filtration chromatographic column. The establishment of the method not only greatly saves precious plasma resources, but also greatly promotes the development of relevant research on pathogenesis and treatment of ATTR diseases.

In order to achieve the technical effects, the invention specifically provides the following technical scheme:

in a first aspect of the present invention, there is provided a method for cloning, expressing, isolating and purifying transthyretin (TTR), the method comprising the steps of:

(1) cloning of TTR gene:

NdeI-TTR-Xho I sequence is designed and PCR amplified through splicing chemically synthesized and purified TTR gene vector. Preferably, the nucleotide sequence of the Nde I-TTR-Xho I sequence is shown in SEQ ID NO. 1.

(2) Construction of recombinant expression plasmids:

digesting the expression vector by using restriction enzyme, inserting the TTR coding sequence into the corresponding site of the vector to obtain the recombinant expression plasmid.

(3) Expression and purification of TTR protein:

culturing competent BL21(DE3) transferred into recombinant expression plasmid at 37 deg.C, adding IPTG to induce protein expression, performing ultrasonic cell disruption, salting out and dialysis, and purifying TTR protein with strong anion exchange chromatography column and gel filtration chromatography column.

In one embodiment, in step (1), the cleavage sites at the N-terminus and C-terminus are NdeI and Xho I, respectively.

In one embodiment, the chemically synthesized TTR gene fragment is 396bp in length.

In one embodiment, in step (2), the expression vector is pET-29b (+).

In one embodiment, in step (3), the concentration of IPTG is 0.2-2 mM.

In one embodiment, in step (3), the conditions for ultrasonic cell disruption are 100-200W, 1s, and 5 s; the bacterial lysate was 50mM Tris, 150mM NaCl, pH 7.5.

In one embodiment, in step (3), the agent used for salting out is ammonium sulfate.

In one embodiment, in step (3), the conditions of the strong anion exchange chromatography column and the buffer are as follows:

a chromatographic column: 50mL Source 15Q.

Anion exchange buffer a: 25mM Tris, 1mM EDTA, pH 8.0.

Elution buffer B: 350mM NaCl.

In one embodiment, in step (3), the conditions for purifying TTR protein by strong anion exchange chromatography are: washing the chromatographic column by 50mL of solution A; solution B was eluted with a linear gradient (160mL, 50-350mM) followed by washing with solution B.

In one embodiment, in step (3), the gel filtration chromatography column and the buffer are under the following conditions:

a chromatographic column: 120mL Superdex 200.

SEC buffer: 10mM sodium phosphate, 100mM KCl, 1mM EDTA, pH 7.4.

In one embodiment, in step (3), the conditions for purifying TTR protein by gel filtration chromatography are: elution flow rate was 1mL/min, SECbuffer elution volume 120 mL.

Compared with the prior art, the invention has the following beneficial technical effects:

(1) establishing a prokaryotic gene expression system capable of being used for expressing TTR protein in a large quantity;

(2) by utilizing the salting-out characteristic of the protein, the foreign protein is greatly reduced through twice precipitation purification;

(3) based on the AKTA protein purification system, the method is simple and convenient to operate, simultaneously realizes multiple steps of automatic washing, online monitoring, real-time observation and the like of the sample, and can adjust the purification process in time according to the sample condition and the subsequent requirement on the purified sample.

Drawings

FIG. 1 Strong anion exchange chromatography purification of TTR profile;

FIG. 2 gel filtration chromatography purification of TTR profile;

FIG. 3 is a graph showing the electrophoresis results of purified TTR protein; wherein, from left to right, thechannel 1 is a protein Marker; thechannel 2 is a sample after strong anion exchange chromatography purification; thechannel 3 is a sample after gel filtration chromatography purification;

FIG. 4UPLC purity test;

FIG. 5 molecular weight size of TTR by mass spectrometry;

FIG. 6 is a graphical representation of the results of tetrameric TTR protein activity.

Detailed Description

The invention will now be further described with reference to the following examples, which are intended to be illustrative of the invention and are not to be construed as limiting the invention.

The experimental procedures used in the following examples are conventional ones without specific mention.

The test consumables used in the examples described below were commercially available without specific reference.

Example 1: construction of recombinant cloning vector Nde I-TTR-Xho I

Primer design for TTR

Using pET-29b (+) as a template, designing an NdeI-TTR-Xho I sequence by splicing a chemically synthesized and purified TTR gene vector, and performing PCR amplification, wherein the nucleotide sequence of the NdeI-TTR-Xho I sequence is shown in SEQ ID NO:1 and consists of an endonuclease site NdeI-TTR protein coding sequence-an endonuclease site Xho I.

NdeI-TTR-Xho I is cloned and connected with digested pET-29b (+) plasmid template

mu.L of Nde I-TTR-Xho I was taken, 2. mu.L of digested pET-29b (+) plasmid template was added, 5. mu.L of ligation buffer and 2. mu.L of sterile water were added to 10. mu.L of the system, and ligation was performed at 16 ℃ for 2 hours.

Cloning and transformation of pET-29b (+) -TTR plasmid template

Transferring 2 mu L of the ligation product to 50 mu LDH5 alpha competent cells, and standing for 30min on ice; heat shock is carried out for 45s at 42 ℃; standing on ice for 5 min; adding 450 μ L LB (Tryptone 10g/L, Yeast extract 5g/L, NaCl 10g/L, pH 7.5), shaking at 37 deg.C and 220rpm for 1 h; centrifuging at 4000rpm for 3 min; most of the supernatant was removed in a clean bench, the pellet was gently suspended, and plated on LB plate containing 50. mu.g/mL kanamycin (Kan); the LB plate was placed upside down in a 37 ℃ incubator overnight.

Identification of clone pET-29b (+) -TTR

Randomly selecting 4 positive clones, and inoculating the positive clones into 4mL LB (Kan +); shaking overnight at 220rpm with a shaker at 37 ℃; plasmid pET-29b (+) -TTR was extracted, stored at-80 ℃ and plasmid sequencing was performed with primer T7.

Example 2: construction of recombinant expression plasmid pET-29b (+) -TTR

1. Digestion and recovery

Nde I and Xho I restriction sites are added into the TTR gene sequence, Nde I-TTR-Xho I and pET-29b (+) plasmid are subjected to double digestion by using Nde I and Xho I, electrophoresis is carried out by using 1% agarose gel, and the target fragment is recovered by using gel.

T4 DNA ligase ligation and transformation

The connecting system is as follows: mu.L of 10 Xligase buffer, 1. mu.L of 10-double-digested pET-29b (+) plasmid, 7. mu.L of Nde I-TTR-Xho I fragment, 1. mu. L T4 DNA ligase, were ligated overnight at 16 ℃.

The ligation products were transformed into DH 5. alpha. competence, transformed, plated on LB plates containing 50. mu.g/mL kanamycin (Kan), and cultured overnight at 37 ℃ in an inverted state.

3. Positive clone identification

Randomly selecting 4 positive clones, and inoculating the positive clones into 4mL LB (Kan +); shaking overnight at 220rpm with a shaker at 37 ℃; the plasmids were extracted for T7 primer-directed plasmid sequencing.

Example 3: inducible expression of TTR protein

1. Materials and reagents

Kanamycin: biyun Tian, China

Isopropyl thiogalactoside: biyun Tian, China

2. Instruments and devices:

an incubator: thermo, USA

UV-5100 ultraviolet visible light breadth meter: shanghai non-analytic Instrument Co., Ltd, China

3. The experimental steps are as follows:

(1) recombinant plasmid transformed Escherichia coli

The recombinant plasmid pET-29b (+) -TTR was transformed into competent BL21(DE3), spread on LB plate containing kanamycin (Kan), and cultured overnight at 37 ℃ in an inverted state.

(2) Isopropyl thiogalactoside (IPTG) induced protein expression

Selecting a single colony of the recombinant strain, inoculating the single colony in LB (Kan +), culturing overnight by a shaking table at 37 ℃ and at 220 rpm; inoculating 2L LB (Kan +) at a ratio of 1:20-40, shaking at 37 deg.C and culturing at 220rpm to OD_600nmWhen 0.6-0.8 was reached, IPTG (0.2-2mM) was added to induce protein expression.

Example 4: purification and identification of TTR protein

1. Materials and reagents

Tris (Tris): biyun Tian, China

EDTA: biyun Tian, China

NaCl: xiong science Inc., China

Coomassie brilliant blue G250: biyun Tian, China

2. Instruments and devices:

a pH meter: MettlerToledo, Switzerland

Low-temperature freezing centrifuge: thermo, USA

Ultrasonic cell crusher: ningbo Xinzhi Biotech GmbH, China

A magnetic stirrer: darongxing laboratory instruments Ltd, China

Protein purification instrument: AKTA, USA

Electrophoresis apparatus: beijing, six Biotechnology Ltd, China

AB Sciex 5800MALDI-TOF/TOF^TMAppearance: AB SCIEX, USA

A multifunctional microplate reader: tecan, Switzerland

Inducible expression of TTR protein

Selecting a single colony of the recombinant strain, inoculating the single colony in LB (Kan +), culturing overnight by a shaking table at 37 ℃ and at 220 rpm; inoculating 2L LB (Kan +) at a ratio of 1:20-40, shaking at 37 deg.C and culturing at 220rpm to OD_600nmWhen 0.6-0.8 was reached, IPTG (0.2-2mM) was added to induce protein expression.

Centrifugation is carried out for 30min at 4 ℃ and 4000g, the supernatant is discarded, and the precipitate is suspended by bacterial lysate.

4. Cell disruption, salting out and dialysis

And (3) fully cracking the bacterial liquid by ultrasonic waves, and then centrifuging the crushed bacterial liquid in a centrifuge at the rotation speed of 16000g for 30min at 4 ℃. The precipitate was discarded and the supernatant was salted out with ammonium sulfate. Finally, the pellet after centrifugation was suspended in 40mL of anion exchange buffer A (25mM Tris, 1mM EDTA, pH 8.0) and dialyzed against buffer A overnight at 4 ℃.

5. Purification of TTR by strong anion exchange chromatography

50mL Source 15Q Strong anion exchange chromatography: solution A (25mM Tris, 1mM EDTA, pH 8.0), solution B (350mM NaCl); after the purified fraction is pumped into the column, it is first subjected to AKTA^TMThe column was washed off non-specifically adsorbed contaminating proteins by washing the column with 50mL of solution A on a fastprotein chromatograph purifactor 100 system, which was then programmed to perform a linear gradient elution with solution B (160mL, 50-350mM) followed by a wash with solution B (50mL, 350 mM). The eluting peak contains TTR, collecting absorption peak II, i.e. about 25mL of TTR eluent, discarding peak I, and the map result is shown in figure 1. Samples were taken for SDS-PAGE and the results are shown in FIG. 3.

6. Purification of TTR by gel filtration chromatography

120mL Superdex 200 gel filtration chromatography column: SEC buffer (10mM sodium phosphate, 100mM KCl, 1mM EDTA, pH 7.4); the purification conditions were: and (3) after the component to be purified is pumped into a chromatographic column, eluting at the flow rate of 1mL/min by using SEC (SEC-eluting) buffer solution of 120mL, and collecting the component with the largest absorption peak of 15mL to obtain high-purity TTR, wherein the spectrum result is shown in figure 2. Sampling and performing SDS-PAGE, and a result shown in figure 3 shows that the purity of the TTR protein is further improved after the purification by gel filtration chromatography; wherein, from left to right, thechannel 1 is a protein Marker; thechannel 2 is a sample after strong anion exchange chromatography purification; thechannel 3 is a sample after gel filtration chromatography purification.

7. Identification of purity of purified TTR

SDS-PAGE gel electrophoresis through laser gray scanner determination of its purity to reach 99% and above, and show that purified TTR protein monomer molecular mass is about 14kDa, the result is shown in figure 3. In addition, the purity identification result of UPLC is also shown as a single peak, the result is shown in fig. 4, and the purity of TTR protein can be up to 99.5% by calculating the percentage of peak area.

Molecular weight size analysis of TTR

The TTR protein end product obtained in this example was desalted and then passed through AB Sciex 5800MALDI-TOF/TOF^TMMass spectrometer detection to determine the molecular weight showed that the molecular weight of the purified TTR protein was about 14kDa as shown in FIG. 5. Consistent with the results of the molecular weight of the monomeric TTR on SDS-PAGE.

9. Activity assay of purified TTR protein

To perform activity analysis on purified TTR protein, the final product TTR protein was first prepared as solutions of different concentration gradients, i.e. 0, 0.5, 1, 2.5, 5, 10 μ M, to which a quantitative amount of fluorescent small molecule a2(S- (4-fluorophenyl) (E) -3- (dimethylamino) -5- (4-hydroxy-3, 5-dimethyltryptyl) thionate) bound only to tetrameric TTR was added, respectively [ Choi S, one DS, Kelly JW. "organic thin selective to transthytin in cells and remains dark units a chemical reaction to yield fluorescent antibody fluorescent protein complex" j.am. chem.2010; 132:16043-16051]Incubation was performed followed by monitoring the fluorescence of the a2 modified TTR conjugate using excitation light at 328nm and emission light at 430nm, with 3 duplicate wells per concentration. And finally, establishing a linear regression model by taking the tetramer TTR concentration as an abscissa and the TTR conjugate fluorescence intensity as an ordinate. The results show that all points fall on the linear regression trend line, and R²0.9989, and the two have higher linear relation, which indicates that the purified tetrameric TTR protein has extremely high activity.

It should be understood that the above-mentioned embodiments are merely preferred embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

SEQ ID NO:1

CATATGGGTCCTACGGGCACCGGTGAATCCAAGTGTCCTCTGATGGTCAAAGTTCTAGATGCTGTCCGAGGCAGTCCTGCCATCAATGTGGCCGTGCATGTGTTCAGAAAGGCTGCTGATGACACCTGGGAGCCATTTGCCTCTGGGAAAACCAGTGAGTCTGGAGAGCTGCATGGGCTCACAACTGAGGAGGAATTTGTAGAAGGGATATACAAAGTGGAAATAGACACCAAATCTTACTGGAAGGCACTTGGCATCTCCCCATTCCATGAGCATGCAGAGGTGGTATTCACAGCCAACGACTCCGGCCCCCGCCGCTACACCATTGCCGCCCTGCTGAGCCCCTACTCCTATTCCACCACGGCTGTCGTCACCAATCCCAAGGAATGACTCGAG

Sequence listing

<110> Dalian run-on Kangtai medical laboratory Co., Ltd

<120> method for cloning, expressing and purifying transthyretin gene

<130> CP211042

<141> 2021-11-16

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 396

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

catatgggtc ctacgggcac cggtgaatcc aagtgtcctc tgatggtcaa agttctagat 60

gctgtccgag gcagtcctgc catcaatgtg gccgtgcatg tgttcagaaa ggctgctgat 120

gacacctggg agccatttgc ctctgggaaa accagtgagt ctggagagct gcatgggctc 180

acaactgagg aggaatttgt agaagggata tacaaagtgg aaatagacac caaatcttac 240

tggaaggcac ttggcatctc cccattccat gagcatgcag aggtggtatt cacagccaac 300

gactccggcc cccgccgcta caccattgcc gccctgctga gcccctactc ctattccacc 360

acggctgtcg tcaccaatcc caaggaatga ctcgag 396

Claims (10)

1. A transthyretin gene (TTR) cloning, expression and protein purification method is characterized by comprising the following steps:

(1) cloning of TTR gene:

NdeI-TTR-Xho I sequence is designed and PCR amplified through splicing chemically synthesized and purified TTR gene vector.

(2) Construction of recombinant expression plasmids:

digesting the expression vector by using restriction enzyme, inserting the TTR coding sequence into the corresponding site of the vector to obtain the recombinant expression plasmid.

(3) Expression and purification of TTR protein:

BL21(DE3) competent Escherichia coli transferred with the recombinant expression plasmid is cultured at 37 ℃, IPTG is added to induce protein expression, then ultrasonic cell disruption, salting out and dialysis are carried out, and TTR protein is purified by a strong anion exchange chromatographic column and a gel filtration chromatographic column.

2. The method according to claim 1, wherein the Nde I-TTR-Xho I sequence is as shown in SEQ ID NO 1.

3. The method of claim 1, wherein in step (2), the vector is pET-29b (+).

4. The method of claim 1, wherein in step (3), the concentration of IPTG is 0.2-2 mM.

5. The method as claimed in claim 1, wherein in the step (3), the conditions for the ultrasonic cell disruption are 100-200W, 1s and 5 s; the bacterial lysate was 50mM Tris, 150mM NaCl, pH 7.5.

6. The method according to claim 1, wherein in the step (3), the agent for salting out is ammonium sulfate.

7. The method of claim 1, wherein the strong anion exchange chromatography column and the buffer are under the following conditions:

a chromatographic column: 50mL Source 15Q;

anion exchange buffer a: 25mM Tris, 1mM EDTA, pH 8.0.

Elution buffer B: 350mM NaCl.

8. The method of claim 1, wherein in step (3), the conditions for purifying TTR protein by strong anion exchange chromatography are as follows: washing the chromatographic column by 50mL of solution A; and (3) carrying out linear gradient elution on the solution B under the elution condition of 160mL and 50-350mM, and then washing with the solution B.

9. The method of claim 1, wherein the gel filtration chromatography column and buffer conditions are as follows:

a chromatographic column: 120mL Superdex 200.

Buffer solution: 10mM sodium phosphate, 100mM KCl, 1mM EDTA, pH 7.4, the buffer is SEC buffer.

10. The method of claim 1, wherein in step (3), the conditions for purifying TTR protein by gel filtration chromatography are as follows: elution flow rate was 1mL/min, SEC buffer elution volume 120 mL.

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