CN114854715A - Metallophosphoesterase, vector, storage solution and preparation method thereof - Google Patents

Metallophosphoesterase, vector, storage solution and preparation method thereof Download PDF

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CN114854715A
CN114854715A CN202210121731.6A CN202210121731A CN114854715A CN 114854715 A CN114854715 A CN 114854715A CN 202210121731 A CN202210121731 A CN 202210121731A CN 114854715 A CN114854715 A CN 114854715A
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acid sequence
amino acid
phosphatase
seq
expression
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孙正龙
李蕾
杨华
关淼
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Kunming University of Science and Technology
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
    • C—CHEMISTRY; METALLURGY
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    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/04—Phosphoric diester hydrolases (3.1.4)
    • C12Y301/04004—Phospholipase D (3.1.4.4)
    • 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
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    • 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

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Abstract

The invention discloses a metal phosphatase, a carrier, a storage solution and a preparation method thereof, wherein the amino acid sequence of the metal phosphatase is SEQ ID NO: 1 and has the activity of metal phosphatase; or, the nucleic acid sequence is SEQ ID NO: 2 or the encoded amino acid sequence is SEQ ID NO: 1. The preparation process of the metal phosphatase adopts an escherichia coli expression system, can obtain the active metal phosphatase with low cost, high purity, high yield and high stability in the shortest production period through the optimization of expression conditions and purification conditions, and has wide market application prospect. The metallophosphoesterase expressed by recombination can be used for in vitro decomposition of glycerol-oriented or alcohol-oriented phosphodiester bonds, and has wide application prospects in the aspects of biopharmaceuticals, clinical treatment of diseases related to lipoprotein abnormality, hydrolysis of lipoprotein, synthesis of phosphatidic acid, use as an emulsifier in industrial production and the like.

Description

Metallophosphoesterase, vector, storage solution and preparation method thereof
Technical Field
The invention relates to the technical field of molecular biology, in particular to a metal phosphatase, a carrier, a storage solution and a preparation method thereof.
Background
Phospholipases (plases) are lipolytic esterases that break down glycerophospholipids, or molecules containing 2FA (free fatty acids) esterified to glycerol and a polar head group and rich in phosphates binding choline, ethanolamine, serine or inositol. The two best studied metallophosphate hydrolases are alkaline phosphatase and violet acid phosphatase (the alkaline and pure acid phosphatases) which catalyze the cleavage of the phosphate groups in the phosphate monoester. Both enzymes utilize a binuclear metal active center to promote the hydrolysis of phosphate. Phospholipases are classified according to their substrate cleavage site: carboxylate acyl hydrolase (PLA, PLB, PLC and PLD) or lysophospholipase (LPLA). It has been found that PLA, PLB and LPLA can cleave FA at the sn-1 or sn-2 position on glycerol; PLC and PLD are phosphodiester hydrolases that cleave glycerol or alcohol oriented phosphodiester bonds, respectively; PLC releases phosphorylated headgroups and diacylglycerides; PLD cleaves the terminal phosphodiester bond, releasing the head group and phosphatidic acid, and the metallophosphoesterase of the present invention belongs to the PLD type. And PLA is classified as PLA1(EC 3.1.1.32) which hydrolyzes fatty acids at the sn-1 position of the glycerol moiety, or PLA2(3.1.1.4) which removes fatty acids at the sn-2 position, according to the position of hydrolysis of its ester bond. PLB (EC 3.1.1.5) can hydrolyze both acyl groups at the sn-1 or sn-2 position of glycerophospholipids and also exhibit LPLA activity. PLCs (EC 3.1.4.3) and PLDs (EC 3.1.4.4) are phosphodiester hydrolases that cleave glycerol-oriented or alcohol-oriented phosphodiester bonds, respectively. These enzymes hydrolyze sphingomyelin and glycerophospholipids, respectively, and play key roles in different physiological processes, including membrane dynamics, cell signaling, migration, growth, and death.
At present, metal phosphoesterases can be extracted from many microorganisms, and sphingomyelinases and phospholipases of several bacteria are crucial for the virulence of extracellular, facultative or obligate intracellular pathogens, since these enzymes contribute to phagosome escape or prevent phagosome maturation, facilitating tissue colonization, establishment and progression of infection, or escape of the immune response. PLase hydrolyzed PL also has potential use in the food industry, particularly in egg yolk for making mayonnaise, refined vegetable oils, and baking and cheese making (De Maria et al, 2007; Borrelli and Trono, 2015). The baking industry has the potential to replace emulsifiers with PLase, as natural emulsifiers can be produced from the hydrolysis of PL and lipids in the dough to improve softness and increase bread volume. This stabilization of the hydrolysate is due to the interaction between the protein and the air bubbles in the dough (Gerits et al, 2014). The metal phosphatase has application potential in various fields of biological pharmacy, clinical medicine, food and the like.
Prokaryotic expression is a biological technology that foreign target genes are constructed into expression plasmid vectors through experimental means such as molecular cloning technology, seamless connection and the like, the expression plasmids are poured into proper expression strains, a specific induction means is utilized to induce a large amount of target proteins to express, and then separation and purification are carried out to obtain the target proteins. Prokaryotic expression combines various molecular biological means including gene engineering, protein engineering and the like, and has the advantages of short period, simple and convenient operation and low cost. However, no scheme for stably expressing metallophosphoesterase by using a prokaryotic expression system is available.
Disclosure of Invention
In order to solve the problems, the invention provides a metallophosphatase, a vector, a storage solution and a preparation method thereof.
According to one aspect of the present invention, there is provided a metallophosphatase comprising the nucleotide sequence shown in SEQ ID No.1, restriction sites NdeI and PstI and 6 XHis Tag at the carboxy terminus of the recombinant protein.
The invention also provides a prokaryotic expression vector of the recombinant metallophosphatase, which comprises the expression gene of the metallophosphatase.
Further, the prokaryotic expression vector is pMAL-c 5X.
The construction method of the prokaryotic expression vector comprises the following steps:
the present invention provides an active metallophosphatase characterized by the following (a) or (b):
(a) the amino acid sequence is SEQ ID NO: 1 and has the activity of metal phosphatase;
(b) the nucleic acid sequence is SEQ ID NO: 2 or a nucleic acid sequence encoding the amino acid sequence of (a);
the invention provides pMAL-c5X series vectors for protein expression, which contain the gene nucleic acid sequence or the expressed protein product containing the amino acid sequence.
The present invention provides strains of E.coli for protein expression, including but not limited to BL21(DE3) and
rosetta (DE3), characterized in that it contains the vector described above.
The present invention provides strains of E.coli for protein expression, including but not limited to BL21(DE3) and Rosetta (DE3), which express protein products comprising the amino acid sequences described above.
The present invention provides restriction enzyme sites used in the construction of metallophosphoesterase expression vectors, including but not limited to: NdeI and PstI.
The method is characterized in that a molecular cloning means is utilized to add a purification Tag at the carboxyl terminal of the metal phosphatase, and the added purification Tag comprises but is not limited to 6 XHis Tag and MBP Tag, and also belongs to the protection scope of the invention.
The expression conditions of the metal phosphatase, including but not limited to the concentration of the bacteria, the concentration of the inducer, the time of the inducible expression and the rotation speed of the shaking table, also belong to the protection scope of the present invention.
The present invention provides a process for purifying metallophosphatases, including but not limited to:
(1) cracking the thallus by an ultrasonic crushing method or a pressure method;
(2) centrifuging to remove cell debris;
(3) affinity chromatography;
(4) ultrafiltration and dialysis.
The invention also discloses a buffer solution formula used in the purification process of the metallophosphatase, and belongs to the protection scope of the invention.
The invention also discloses a storage liquid formula and storage conditions of the finished product of the metal phosphatase, and belongs to the protection scope of the invention.
The translated full-length protein has 285 amino acid residues, wherein 21 amino acids at the amino terminal are signal peptide sequences, and the signal peptide is cut off when the protein is secreted outside cells through a membrane. The mature enzyme molecule contains 264 amino acid residues, has a molecular weight of 33.65kD and an isoelectric point of 6.75.
The invention has the beneficial effects that:
the preparation process of the metal phosphatase adopts an escherichia coli expression system, can obtain the active metal phosphatase with low cost, high purity, high yield and high stability in the shortest production period through the optimization of expression conditions and purification conditions, and has wide market application prospect. The metallophosphoesterase expressed by recombination can be used for in vitro decomposition of glycerol-oriented or alcohol-oriented phosphodiester bonds, and has wide application prospects in the aspects of biopharmaceuticals, clinical treatment of diseases related to lipoprotein abnormality, hydrolysis of lipoprotein, synthesis of phosphatidic acid, use as an emulsifier in industrial production and the like.
Drawings
FIG. 1 is an SDS-PAGE electrophoresis chart of the prokaryotic expression vector pMAL-c5X-Amuc _1901 of the recombinant protein of the metallophosphatase AMUC _1901 in the Escherichia coli induced expression:
lane M is a protein molecular weight standard; lane 1 is uninduced by E.coli; lane 2 is the induction of expression vector pMAL-c5X-Amuc _ 1901;
FIG. 2 SDS PAGE of BSA concentration quantification of Amuc _1901 recombinant enzyme purified on Ni column
Lane M is a protein molecular weight standard; lane 1 ═ 0.05mg/ml BSA; lane 2 ═ 0.1mg/ml BSA; lane 3 ═ 0.2mg/ml BSA; lane 4 ═ 0.3mg/ml BSA; lane 5 ═ Amuc _1901 recombinase.
FIG. 3 is a standard curve method for measuring the enzyme activity of recombinant phosphatase, wherein p-NA (p-nitrophenylacetate) is used as a substrate, and a standard curve of the activity of metallophosphatase is measured under the conditions of 37 ℃ and pH 8;
FIG. 4 Biochemical Properties of recombinant phosphatase
(A) Substrate preference using p-nitrophenyl phosphate monoester: p-NA ═ p-nitrophenylacetate; p-NB ═ p-nitrobenzyl butyrate; p-NO p-nitrooctylic acid phenyl ester; p-ND ═ p-nitrophenyldodecanoate; p-NP ═ p-nitrophenylpalmitate; p-NS ═ p-nitrophenylstearate. (B) Influence of pH;
FIG. 5 Biochemical Properties of recombinant phosphatase
(A) The temperature influence. (B) The effect of monovalent and divalent ions, detergents, reducing agents and metal ion chelators, control-no reagent treated samples;
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention provides an expression gene of recombinant metal phosphatase AMUC _1901, which comprises a nucleotide sequence shown as SEQ ID No.2, restriction enzyme sites NdeI and PstI and 6 × His Tag positioned at the carboxyl terminal of a recombinant protein.
The invention also provides a prokaryotic expression vector of the recombinant metal phosphatase AMUC _1901, which comprises the expression gene. In a preferred embodiment, the prokaryotic expression vector selected is pMAL-c 5X.
The invention also provides a recombinant fusion protein AMUC _1901, the amino acid sequence of the recombinant fusion protein AMUC _1901 is shown in SEQ ID No.1, and the nucleotide sequence coding amino acid sequence shown in SEQ ID No.2 is shown in SEQ ID No.1, and the recombinant fusion protein AMUC _1901 is provided. The invention further provides a preparation method of the recombinant fusion protein AMUC _ 1901.
The present invention will be further described with reference to the following examples.
Example 1 preparation method of recombinant metallophosphatase AMUC _1901, expression Gene, recombinant expression vector, and vector construction method thereof
The species sources of the metallophosphoesterases of this example are: akkermansia mucina ATCC BAA-835B2UNJ9
Uniprot No.: b2UNJ9_ AKKM 8. The amino acid sequence of the metal phosphatase of the present embodiment is shown in SEQ ID No.1, and the nucleotide sequence of the gene encoding the metal phosphatase is shown in SEQ ID No. 2.
In this example, the amino-terminal signal peptide sequence was removed by artificially synthesizing the full-length gene sequence, and ligated into the vector pMAL-c5X, with NdeI and PstI cleavage sites. Amuc _1901 gene was cloned from AKK genome using Amuc _1901 gene specific primers, and PCR was performed using high fidelity enzyme available from NEB.
The plasmid vector pMAL-c5X-Amuc _1901 was transformed into E.coli Rosetta (DE3) competent cells by chemical method, and positive transformants were selected by using a petri dish containing 50. mu.g/ml kanamycin solid LB medium to obtain a metallophosphatase-expressing strain. As shown in FIG. 1, the induction results were examined by polyacrylamide gel electrophoresis for strains expressing metallophosphoesterases.
As an alternative embodiment, the Escherichia coli strain used for transformation in this example may be BL21(DE 3).
EXAMPLE 2 expression of metallophosphatases
The positive clones expressing metallophosphatase obtained by transformation were picked, and the recombinant expression strains were inoculated in 5ml of liquid ampicillin (LB) medium and cultured with shaking at 37 ℃ and 220rpm for 5-6 hours.
The strain culture broth was transferred to 200ml of LB liquid medium, and after shaking culture at 37 ℃ and 220rpm for 6 hours, the strain was cultured until OD was 0.6-0.8. The inducer IPTG was added to a final concentration of 0.5mM, and the mixture was cultured overnight at 16 ℃ with shaking at 180 rpm. Collecting thallus, rotating at 8000rpm, and centrifuging for 15 min. Resuspending the bacterial pellet with 50mM Tris-HCl pH8.0 and 200mM NaCl, lysing for 10min at 850MPa-900MPa by a homogenizer, centrifuging at high speed for 20min, and taking the supernatant. Filtering with 0.45um filter membrane, loading the cracked supernatant into Ni affinity chromatography column, incubating at 4 deg.C for 30min, eluting with buffer solution containing 20mM imidazole, eluting with eluent containing 300mM imidazole, ultrafiltering, concentrating, changing liquid, packaging to obtain metal phosphatase finished product, adding storage solution, and storing at-20 deg.C.
Example 3 measurement of the Activity of metallophospholipases and Biochemical index verification
The metallophosphatases prepared in example 2 were stored in a storage solution of metallophosphatases, the formulation of which was: 1xPBS buffer solution (Na2HPO41.42g, KCl0.2g, NaCl 8g and KH2PO40.27g, all the components are weighed in a 1L beaker, 800mL of deionized water is added into the beaker, a glass rod is stirred and dissolved, the volume is determined to be 1L, NaOH or dilute hydrochloric acid is added to adjust the pH value to 7.4), and 50% glycerol is added to obtain the total 10mL of metal phosphatase finished product.
Taking p-nitrophenyl phosphate monoester as a substrate, breaking a phosphodiester bond between a PNP (p-nitrophenyl) head group and phosphate ester by using metal phosphatase to release the PNP, wherein the PNP has a maximum absorption peak at OD410, and drawing a PNP standard curve to detect an absorbance value of the PNP, wherein the rate of hydrolyzing the p-nitrophenyl phosphate monoester by AMUC _1901 can be reflected. The reaction system is as follows:
8.89mM PNP phosphate monoester substrate 18ul
0.2mg/ml AMUC _1901 recombinase 40ul
Reaction buffer 162ul
OD410 was measured at 37 ℃ for 30min after the above reaction in a 96-well plate, and 1U unit was defined as the amount of enzyme corresponding to the release of 1. mu. mol of p-nitrophenol (PNP) per minute at 37 ℃. The enzyme activity of the recombinant metal phosphatase is 299.86U/L and the enzyme activity concentration of the recombinant finished product is 30U/ml by using a standard curve method.
The concentration of the recombinant phosphatase was determined by the BCA method to obtain a concentration of the metallophosphatase of 0.25mg/ml, i.e., the specific activity of the recombinant enzyme was 120U/mg.
FIG. 3 is a graph showing the standard enzyme activity of the finished metallophosphoesterase prepared according to the example of the present invention.
The preparation process of the metal phosphatase adopts an escherichia coli expression system, can obtain the active metal phosphatase with low cost, high purity, high yield and high stability in the shortest production period through the optimization of expression conditions and purification conditions, and has wide market application prospect. The metallophosphoesterase expressed through recombination can be used for in vitro decomposition of glycerol-oriented or alcohol-oriented phosphodiester bonds, and has wide application prospects in the aspects of biopharmaceuticals, clinical treatment of diseases related to lipoprotein abnormality, hydrolysis of lipoproteins, synthesis of phosphatidic acid, use as an emulsifier in industrial production and the like.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
<110> university of Kunming science
<120> metallophosphoesterase, and vector, storage solution and preparation method thereof
<160> 2
<210> 1
<211> 264
<212> Protein
<213> Artificial Sequence (Artificial Sequence)
<400> 1
MIGFSLFMWAWQVEPRRVETVTAAMEMPQWKRKESPPLRIAIAGDFHLRPNGGDLAHRYMETIMEARPDMIFLLGDYANGHTRESSMAPETAREYFKMLKAPLGIFAVQGNHDQYYGWNLWRNMFSGLGILPMWNDSLLLHLPGGRELQLSSVRDDYHLRIRPEELPLRFSPDIPHILLS
HVPDIFPLLAPGTADLVISAHTHGGQICLPGGRALANISREKVKFSYPWSQLNGTPFLITRGLGCSVLPLRFCCPPEIIVLEIQ
<210> 2
<211> 858
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
ATGAAGTCACGACCACGCCGCCCCTTTCTCAACAAATTGGCAATTATCTTTGTCCCAATATTGATGATCGGCTTCTCCTTATTCATGTGGGCATGGCAGGTGGAACCCAGGCGTGTGGAAACCGTCACGGCTGCGATGGAGATGCCTCAGTGGAAGAGAAAAGAAAGCCCTCCCCTCCGTATCGCGATTGCCGGGGATTTCCATCTGCGTCCGAACGGGGGCGATCTGGCGCACAGGTACATGGAAACAATCATGGAAGCTCGGCCGGATATGATTTTCCTGCTGGGGGATTATGCCAACGGCCACACGAGGGAGAGCAGCATGGCCCCGGAAACGGCCCGGGAATATTTCAAGATGTTGAAAGCCCCTCTCGGCATTTTTGCCGTGCAGGGCAACCATGACCAGTATTACGGCTGGAACTTATGGCGCAACATGTTTTCCGGGCTGGGCATCCTGCCCATGTGGAATGATTCCCTGCTGCTGCACCTTCCCGGAGGCCGTGAGCTGCAGCTCTCTTCCGTCAGGGACGACTACCACCTGAGAATCAGGCCGGAGGAATTGCCTCTGCGTTTTTCCCCGGATATTCCGCATATCCTTCTTTCACATGTACCCGACATTTTTCCTCTGCTGGCTCCCGGCACGGCGGACCTCGTCATCAGCGCGCATACCCACGGAGGCCAGATATGCCTTCCCGGAGGCAGGGCCCTGGCAAACATCTCACGAGAGAAGGTCAAATTTTCCTATCCCTGGTCCCAGCTGAACGGAACCCCCTTCCTCATTACCCGCGGTTTGGGATGCAGCGTTCTTCCCCTGCGCTTCTGCTGCCCTCCGGAAATCATCGTGCTGGAAATCCAATAA

Claims (10)

1. A metallophosphatase comprising an amino acid sequence of SEQ ID NO: 1, or an active fragment, an analogue or a derivative of the protein with site-directed mutation or modification of amino acid, and has the activity of metal phosphatase.
2. The phosphatase according to claim 1, wherein the amino acid sequence of the polypeptide of the amino acid sequence or of the active fragment, analogue or derivative thereof, which is mutated or modified at the amino acid site, has a sequence identical to the sequence of SEQ ID NO: 2 or the encoded amino acid sequence is SEQ ID NO: 1 is at least 95% similar.
3. An isolated phosphatase characterized by the following (a) or (b) or (c) or (d):
(a) the amino acid sequence is SEQ ID NO: 1 and has the activity of metal phosphatase;
(b) the amino acid sequence of the polypeptide has 70 percent of homology with (a) and above;
(c) the nucleic acid sequence is SEQ ID NO: 2 or a nucleic acid sequence encoding the amino acid sequence of (a);
(d) the nucleic acid sequence encodes an amino acid sequence which has 70% or more homology with (a).
4. A suitable expression vector comprising the nucleic acid sequence of the gene of claim 1 or the expressed protein product comprising the amino acid sequence of claim 2.
5. Escherichia coli strain for protein expression, characterized in that it contains the vector of claim 4 or the expressed protein product contains the amino acid sequence of claims 1, 2.
6. A restriction endonuclease site for constructing a metallophosphatase expression vector, comprising NdeI and a restriction endonuclease site PstI.
7. The method of claim 5, wherein the carboxyl-terminal of the metallophosphatase is tagged with a purification tag, including but not limited to 6 XHis tag and MBP tag, by molecular cloning.
8. The expression condition of metallophosphoesterase according to claim 1, comprising bacterial cell concentration at the time of induction expression, inducer concentration, time of induction expression, and shaking table rotation speed.
9. A method for purifying metallophosphatase, comprising the steps of:
s1, cracking the thallus by an ultrasonic crushing method or a pressure method;
s2, centrifuging to remove cell debris;
s3, affinity chromatography;
s4, ultrafiltration and dialysis.
10. A storage solution of finished metal phosphatase is characterized in that the preparation method comprises the following steps: 1xPBS buffer plus 50% glycerol.
CN202210121731.6A 2022-02-09 2022-02-09 Metallophosphoesterase, vector, storage solution and preparation method thereof Pending CN114854715A (en)

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