In-vitro expression and purification preparation method of Salsolinol synthetase

Abstract

The invention discloses an in-vitro expression and purification preparation method of Salsolinol synthetase, and belongs to the field of gene engineering and protein expression. The method comprises the following steps: firstly, constructing a Salsolinol synthetase expression vector with a histidine tag (6 * His), the expression vector is pET-28a, and introducing into a host cell to induce expression; a high-purity enzyme is obtained by adopting methods such as affinity chromatography and gel filtration chromatography, and enzyme activity determination is carried out. Based on an amino acid sequence of Salsolinol synthetase, an efficient in-vitro expression vector is constructed, and high-purity enzyme is prepared.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering and protein expression, and specifically relates to the in vitro expression and separation and purification of Salsolinol synthase, and utilizes genetic engineering technology to prepare high-purity enzymes. Background Art

[0002] Catechol isoquinolines (CTIQs) can cause damage to dopaminergic neurons and are considered to be potential endogenous neurotoxins that can cause Parkinson's disease (PD). Salsolinol synthase is the first key enzyme in the synthesis of this type of substance in the brain. It is an enzyme that catalyzes dopamine (DA) and acetaldehyde (Ach) to produce Salsolinol, which directly affects the metabolic process of Sal and its derivatives in the body, and may play an important role as a key factor in the pathogenesis of PD. The research team has previously established a method for separating and purifying Salsolinol synthase from an organism (SD rat brain) and identified its amino acid sequence. However, the method for separating and purifying the enzyme in vivo has a high production cost and a low enzyme recovery rate, which cannot meet the extensive in vitro research on the biological properties of the enzyme. Based on the amino acid sequence of Salsolinol synthase, the present invention constructs an efficient in vitro expression vector and prepares a high-purity enzyme, providing a basis for subsequent studies on the enzyme properties of this enzyme. Summary of the invention

[0003] The technical problem solved by the invention is to provide a construction method of an in vitro carrier of Salsolinol synthase and a purification preparation method thereof.

[0004] The invention first constructs a Salsolinol synthase expression vector with a histidine tag (6×His), the expression vector is pET-28a, and introduces it into a host cell to induce expression; uses affinity chromatography, gel filtration chromatography and other methods to obtain a high-purity enzyme, and performs enzyme activity determination.

[0005] The specific contents are as follows:

[0006] (1) Construction of Salsolinol synthase expression vector

[0007] The research group obtained the Salsolinol synthase DNA sequence by point mutation in the early stage. The target sequence was amplified and connected with the expression vector pET-28a plasmid. DNA sequencing confirmed that two expression vectors of the Salsolinol synthase gene, pET-28a-His-Salsyn and pET-28a-His-Salsyn-His, were constructed.

[0008] The nucleotide sequence of the His-Salsyn is:

[0009] CATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCGCAGCCATATGGCTAGCATGACTGGTGACAGCAAATGGGTCGCGGATCCGAATTCATGCAAATCTTCGTGAAGACCCTGACCGGCAAGACCATCACCCTGGAGGTGGAGCCCAGTGACACCATCAAGAACGTGAAGGCCAAGATCCAGGATAAGGAGGGCATCCCCCCTGACCAGCAGAGGCTCATCTTTGCCGGCAAGCAGCTGGAAGATGGCCGCACCCTCTCTGACTACAACATCCAGAAAAAGTCAACCCTGCACCTAGTCCTCCGCCTGAGGGTTGACTATTAA

[0010] Among them: CATCATCATCATCATCAC is the His tag; AGCAGCGGCCTGGTGCCGCGCGGCGCAGCCATATGGCTAGCATGACTGGTGACAGCAAATGGGTCGCGGATCCGAATTC is the linker and T7 tag; ATGCAAATCTTCGTGAAGACCCTGACCGGCAAGACCATCACCCTGGAGGTGGAGCCCAGTGACACCATCAAGAACGTGAAGGCCAAGATCCAGGATAAGGAGGGCATCCCCCCTGACCAGCAGAGGCTCATCTTTGCCGGCAAGCAGCTGGAAGATGGCCGCACCCTCTCTGACTACAACATCCAGAAAAAGTCAACCCTGCACCTAGTCCTCCGCCTGAGGGTTGACTAT is the Salsolinol synthase gene sequence; TAA is the stop codon.

[0011] The nucleotide sequence of the His-Salsyn-His is as follows:

[0012] CATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCGGCAGCCATATGGCTAGCATGACTGGTGACACGAAATGGGTCGCGGATCCGAATTCATGCAAATCTTCGTGAAGACCCTGACCGGCAAGACCATCACCCTGGAGGTGGAGCCCAGTGACACCATCAAGAACGTGAAGGCCAAGATCCAGGATAAGGAGGGCATCCCCCCTGACCAGCAGAGGCTCATCTTTGCCGGCAAGCAGCTGGAAGATGGCCGCACCCTCTCTGACTACAACATCCAGAAAAAGTCAACCCTGCACCTAGTCCTCCGCCTGAGGGTTGACTATCTCGAGCACCACCACCACCACCAC

[0013] Among them: CATCATCATCATCATCAC is the His tag; AGCAGCGGCCTGGTGCCGCGCGGCGGCAGCCATATGGCTAGCATGACTGGTGACACGAAATGGGTCGCGGATCCGAATTC is the linker and T7 tag; ATGCAAATCTTCGTGAAGACCCTGACCGGCAAGACCATCACCCTGG

[0014] AGGTGGAGCCCAGTGACACCATCAAGAACGTGAAGGCCAAGATCC

[0015] AGGATAAGGAGGGCATCCCCCCTGACCAGCAGAGGCTCATCTTTGC

[0016] CGGCAAGCAGCTGGAAGATGGCCGCACCCTCTCTGACTACAACATC

[0017] CAGAAAAAGTCAACCCTGCACCTAGTCCTCCGCCTGAGGGTTGACTAT is the Salsolinol synthase gene sequence; CTCGAGCACCACCACCACCACCAC is the linker and His tag.

[0018] (2) Inducible expression, isolation and purification of Salsolinol synthase

[0019] The two Salsolinol synthase expression vectors obtained in step (1) are transformed into the expression strain BL21 (DE3), and the transformed single colonies are inoculated in LB medium containing kanamycin resistance. When the OD value of the bacterial solution is 0.6-0.8, IPTG is used to induce and collect the bacteria. Resuspend with Tris Buffer, centrifuge or filter after ultrasonic disruption to obtain a bacterial supernatant, and separate the supernatant with Ni-NTA chromatography column to obtain a crude Salsolinol synthase protein sample; then the crude Salsolinol synthase protein sample is loaded into a gel filtration chromatography column for further purification to obtain a Salsolinol synthase sample.

[0020] (3) Functional identification of salsolinol synthase

[0021] In order to verify whether the purified Salsolinol synthase protein has the corresponding catalytic activity, we designed an experiment to verify its activity based on the enzymatic reaction it can catalyze. Substrates Ach and dopamine DA were added to the system and incubated at 16-37°C for 40 minutes. The reaction product was acid precipitated and then detected by LC / MS, and compared with the Salsolinol standard sample, confirming that the purified Salsolinol synthase has activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 : The plasmid map of pET-28a-His-Salsyn-His with His tags at both the N-terminus and the C-terminus.

[0023] Figure 2 : The plasmid map of pET-28a-His-Salsyn with His tag only at the C-terminus.

[0024] Figure 3 : Affinity chromatography SDS-PAGE electrophoresis results of the eluted fractions with different imidazole concentrations. From left to right, M and 1-7 are numbered as Marker, 300mM imidazole, 100mM imidazole, 50mM imidazole, 20mM imidazole, 0mM imidazole, affinity chromatography effluent and whole protein bacterial solution.

[0025] Figure 4 : Gel filtration chromatography results and SDS-PAGE. From left to right, they are Marker, with DTT, and without DTT. (DTT can destroy the disulfide bonds formed between the amino acid cysteine ​​and destroy the protein structure)

[0026] Enzyme activity function verification. ctrl is the control group with only substrate, H and HH are the purified Salsolinol synthase with His tag added to the substrate at the C-terminus and His tag at both ends, respectively. DETAILED DESCRIPTION

[0027] The present invention is further described below, but the present invention is not limited to the following embodiments.

[0028] 1. Construction of Salsolinol synthase expression plasmid

[0029] The E. coli expression system is a widely used classic protein expression system, with advantages such as clear genetic background, low cost, high expression, and easy product purification. The expression strain E. coli BL21 (DE3) selected in the present invention is one of the most widely used strains in recombinant protein research, and can regulate the expression of target genes downstream of the T7 promoter under the induction of IPTG (isopropyIβ-D-thiogalactoside).

[0030] 1.1 Acquisition of target genes

[0031] The research team previously isolated, purified and identified Salsolinol synthase from the brain of SD rats, and determined that it is a small molecular weight protein similar to the ubiquitin protein acid sequence, containing 77 amino acids (MQIFVKTLTGKTITLEVEPSDTIKNVKAKIQDKEGIPPDQ QRLIFAGKQL EDGRTLSDYN IQKKSTLHLV LRLRVDY). The 24th and 64th amino acids at the N-terminus of ubiquitin are both glutamic acid (E), and the corresponding positions of Salsolinol synthase are both lysine (K); the 75th and 76th amino acids at the N-terminus of ubiquitin are both glycine (G), and the corresponding positions of Salsolinol synthase are valine (V) and aspartic acid (D), respectively. The research team obtained the Salsolinol synthase gene sequence by performing point mutations on four sites of the ubiquitin gene.

[0032] 1.2 Vector construction

[0033] 1.2.1 Double restriction digestion reaction of pET-28a empty vector

[0034] The present invention selects a pET-28a plasmid containing a His-tag before and after an empty vector restriction site, and only constructs an H group at the C-terminus to add a stop codon at the N-terminus of the target gene to interrupt translation.

[0035] Plasmid pET-28a was double-digested with E.coR I and Xho I. The reagents were prepared and mixed on ice, and digested in a metal bath at 37°C for 30 min, with a total volume of 50 μL. The formulation is shown in Table 1-1 below.

[0036] Table 1-1 Enzyme Digestion System

[0037]

[0038] 1.2.2 Agarose gel electrophoresis

[0039] In order to separate the pET-28a plasmid and the remaining nucleic acid after restriction digestion, agarose gel electrophoresis was used for separation.

[0040] Weigh 0.3g agarose into a conical flask, add 30mL 1×TAE, and heat in a microwave oven. Take out the solution after it boils, let it stand for a while, then add 2.5μL DNA marker and shake well. Pour the agarose gel solution into the gel plate and let it stand for 20min. When the gel is completely solidified, pull out the comb vertically, remove the gel plate and remove the excess gel debris, put it into the electrophoresis tank, the electrophoresis solution covers the gel plate, and the loading hole is close to the negative pole of the electrophoresis tank. Set the voltage to 140v, electrophoresis for 30min, and start electrophoresis.

[0041] 1.2.3 Ligation of target gene and double-enzyme-digested plasmid

[0042] Recover the structure from the previous step by DNA gel, then use Nano Drop to detect the nucleic acid concentration. According to the concentration of the target gene and the concentration of the double-enzyme-cut plasmid, mix the target DNA fragment and the double-enzyme-cut plasmid vector fragment at a ratio of 1:3, as shown in Table 1-2. Prepare the connection system on ice, use a pipette to gently mix, and centrifuge briefly to collect the reaction solution to the bottom of the centrifuge tube. Set the connection in a 50℃ metal bath for 15 minutes, and then put it in a 4℃ refrigerator to cool and terminate the reaction.

[0043] Table 1-2 Connection system

[0044]

[0045] The plasmid was transferred into DH5α competent cells and cultured overnight on LB medium containing kanamycin resistance. The next day, a single colony was picked to prepare a monoclonal bacterial solution, and the plasmid was tested by DNA sequencing, and the sequencing results were correct.

[0046] 2. Expression and purification of salsolinol synthase

[0047] His-tag is a tag composed of 6 histidines, which can be inserted at the C-terminus or N-terminus of the target protein. 2+ Affinity binding occurs, and different concentrations (0, 20, 50, 100, 300 mM) of imidazole are used for competitive binding to elute the target protein from the fixed metal ions, thereby achieving the purpose of separation and purification. The affinity chromatography column used in the present invention is prepared by covalently coupling the chelating agent NTA to a 6% (m / V) agarose medium, and then fixing the chelated metal ions at four sites.

[0048] Gel filtration chromatography is a method for separating proteins by taking advantage of the different elution times of protein molecules of different molecular weights when passing through a filler with a certain pore size. The present invention uses a Superdex 200pg gel filtration chromatography column.

[0049] 2.1 Inducible expression of salsolinol synthase

[0050] Take BL21 (DE3) competent cells from -80℃, and divide them into two equal portions after thawing, each with 50μL. Add the two plasmids to the competent E. coli, flick to mix, place at -20℃ for transformation for 30min, heat shock at 42℃ for 70sec, and then cool on ice bricks for 2-3min. Add 900μL of liquid LB medium without resistance to each competent cell, mix well, and shake at 37℃ for 1h. Centrifuge at 5000rpm for 5min to remove 900mL LB medium, use a pipette to blow and mix the remaining liquid, evenly apply kanamycin resistance on the plate, and place it at 37℃ for inversion overnight. The next day, pick a single colony from the culture dish and inoculate it in 1mL of kanamycin resistance LB medium to obtain BL21 monoclonal bacterial liquid.

[0051] Take 20mL of the above monoclonal bacterial solution and inoculate it into 2L of LB medium containing kanamycin. When the OD value is greater than 0.6, add IPTG at a final concentration of 0.2mM and induce overnight at 16℃. The next day, centrifuge the bacterial solution at 4℃ and 6500rpm for 30min, remove the medium, and obtain the bacterial precipitate. For the two proteins with His-tag (HH) introduced at both ends and His-tag (H) introduced only at the C-terminus, mix the bacterial solution and PMSF (benzoyl fluoride) at a ratio of 100:1 (v / v), and use 20mM Na 3 PO 4 , 150mM NaCl, pH=7.4 Tris Buffer. Sonicate the bacteria on ice at 300w, 3s each time, 5s interval, for a total of 30min. Centrifuge at 2300rpm for 60min at 4℃, collect the supernatant and place on ice.

[0052] 2.2 Purification of Salsolinol synthase

[0053] 2.2.1 Affinity chromatography

[0054] The supernatant containing the Salsolinol synthase was filtered using a 0.22 μm filter membrane to remove cell debris. The supernatant was then passed through a Ni-NTA chromatography column using a peristaltic pump at a flow rate of 2 mL per minute to allow the target protein to bind to the chromatography column. After loading, the sample was gradually eluted using a gradient imidazole solution, with the imidazole gradient from low to high being 0 mM, 20 mM, 50 mM, 100 mM, and 300 mM to separate the target protein from the impurities. The eluted sample was tested by SDS-PAGE, and the sample containing the target protein was selected for concentration.

[0055] 2.2.2 Gel filtration chromatography

[0056] The sample purified in the previous step was concentrated to 500 μL volume by centrifugation at 4°C 2300 rpm for 60 min in a 3K ultrafiltration tube, and then loaded onto a Superdex 200 pg gel filtration column through a 500 μL loop for further purification. After adding reducing or non-reducing loading buffer to the sample, it was tested by SDS-PAGE, and the protein molecular weight and aggregation form were estimated based on the elution volume during the purification process and the standard sample curve. The purified target protein sample was collected and concentrated.

[0057] 3. Identification of Salsollinol Synthetase

[0058] In order to detect whether the protein sample obtained by the above method is Salsolinol synthase protein, the present invention adopts enzyme reaction method for detection. The above concentrated sample is obtained and the corresponding protein concentration is detected by BCA quantitative method. According to the concentration obtained by the measurement, the reaction solution is configured according to the system in Table 3-1, the enzymatic system is mixed, incubated at 37°C for 40 minutes in the dark, and placed on ice after the incubation. 10 μL 1M PCA solution is added to each group to terminate the reaction and acid precipitate the protein. After mixing, centrifuge at 12000rpm 4°C for 10 minutes, and take the supernatant on ice in the dark.

[0059] Table 3-1 Enzymatic reaction system

[0060]

[0061] The reaction products were detected by LC / MS and compared with Salsolinol standards to verify the products.

Claims

1. Salsolinol synthase, characterized in that The gene sequence is: MQIFVKTLTG KTITLEVEPSDTIKNVKAKIQDKEGIPPDQ QRLIFAGKQL EDGRRTLSDYNIQKKSTLHLV LRLRVDY.

2. The method for inducing expression and separating and purifying salsolinol synthase, characterized in that: The target sequence was amplified and connected to the expression vector pET-28a plasmid. After identification, two expression vectors pET-28a-His-Salsyn and pET-28a-His-Salsyn-His of the Sslsolinol synthase gene were constructed. (2) Inducible expression and purification of salsolinol synthase The two Salsolinol synthase expression vectors obtained in step (1) are transformed into the expression strain BL21 (DE3), and the transformed single colonies are respectively inoculated in LB medium containing kanamycin resistance. When the OD value of the bacterial solution is 0.6-0.8, IPTG is used to induce and collect the bacteria; Tris Buffer is used to resuspend, ultrasonically disrupt, and then centrifuged or filtered to obtain a bacterial supernatant, and the supernatant is separated by a Ni-NTA chromatography column to obtain a crude Salsolinol synthase protein sample; then the crude Salsolinol synthase protein sample is loaded onto a gel filtration chromatography column for purification to obtain a Salsolinol synthase sample.

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