CN108998462B - Escherichia coli expression system of manganese ion-containing recombinant protein and application method thereof - Google Patents

Abstract

The invention discloses an escherichia coli expression system of manganese ion-containing recombinant protein and an application method thereof. The recombinant expression plasmid in the Escherichia coli expression system is one or the combination of the following situations: (1) at least comprises an escherichia coli molecular chaperone gene and an enzyme protein gene containing manganese ions; (2) comprises an escherichia coli molecular chaperone gene, an enzyme protein gene containing manganese ions and a protein gene related to over-expression or inhibition of manganese ion channels; (3) comprises an escherichia coli molecular chaperone gene, an enzyme protein gene containing manganese ions and a protein gene related to the influence of intracellular manganese ion concentration. Through the construction and optimization of the escherichia coli expression system, the efficient soluble and active expression of manganese ion-containing enzyme protein can be realized, compared with the traditional method, the method has the advantages of high efficiency, simple purification process and low cost, and is beneficial to the industrial production and application of manganese ion-containing enzyme.

Description

Escherichia coli expression system of manganese ion-containing recombinant protein and application method thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an escherichia coli expression system of manganese ion-containing recombinant protein and an application method thereof.

Background

Escherichia coli is the first host bacterium for recombinant protein production, has the advantages of clear genetic background, simple culture operation, high transformation and transduction efficiency, fast growth and reproduction, low cost, capability of fast and large-scale production of target protein and the like, and is always the most widely applied expression system in genetic engineering (crystal generation and the like. research progress of the Escherichia coli expression system. pharmaceutical biotechnology 2005,12(6): 416-420; courgette's republic of China, research progress of the Escherichia coli expression system. university of Yangtze river (natural science edition), 2008,5(3): 77-83). Recent researches show that Escherichia coli expression vectors widely applied in scientific research and industry mainly comprise pGE series, pQE series and pET series, wherein the high-efficiency expression vector widely applied at present is pET, and the system is the expression vector which has the highest efficiency, the highest yield and the highest success rate for expressing foreign proteins in Escherichia coli. It is originally a T7RNA polymerase/promoter system constructed by using exogenous RNA polymerase which is matched with promoter and can effectively transcribe specific gene, and can produce large quantity of target protein from various genes (including prokaryotic cell and eukaryotic cell) (Qihao et al. research progress of colibacillus expression system and yeast expression system. Anhui agricultural science, 2016,44(17): 4-6). There are 11 different DE3 lysogenic host bacteria of the pET system, the most widely used of which is BL21 and its derived strains. The Rosetta host bacterium is derived from BL21, and can enhance the expression of eukaryotic protein with rare codons of Escherichia coli, thereby avoiding expression limitation caused by the codon usage frequency of the Escherichia coli.

Although the E.coli expression system has many advantages as the most widely used protein expression system at present, there are some disadvantages that the expressed protein often forms inclusion bodies or is degraded by protease, and most of the reasons are that the expressed protein is not folded correctly. In addition, the inclusion body protein is difficult to purify, a prokaryotic expression system is imperfect in post-translational processing and modification system, and the biological activity of an expression product is low. At present, the main experimental schemes for improving the soluble expression of target protein in an escherichia coli expression system are as follows: (1) reducing the rate of protein synthesis; (2) changing the culture medium; (3) co-protein expression with a chaperone or foldase. Common molecular chaperones in E.coli are: GroES-GroEL, DnaK-DnaJ-GrpE and ClpB; common folding enzymes used in Escherichia coli include PPI's, DsbA, DsbC and PDI; (4) secretory expression, and the target protein is secreted into the periplasmic space. The periplasmic space has the existence of folding enzymes DsbA and DsbC, which help the protein fold correctly; (5) using a specific strain, such as AD494 or Origami; (6) fusion expression with soluble label protein; (7) in vitro unfolding and refolding.

The most widely used commercial molecular Chaperone Plasmid is Chaperone Plasmid Set of Takara corporation, which contains five types of plasmids, each of which can effectively express different types of Chaperone protein groups, and each Chaperone protein group has synergistic effect and participates in protein folding together. It has been reported that co-expression of a protein of interest with one of the "Chaperone team" increases the recovery of soluble proteins using conventional methods, and that these expressed proteins are often not recoverable due to the formation of inclusion bodies.

The above experimental scheme for promoting the soluble expression of the target protein in the escherichia coli expression system has a promoting effect on the soluble expression of a plurality of proteins forming inclusion bodies in the escherichia coli by using alone or in combination, and different methods applicable to different proteins are different and need to be searched and optimized. For some complex enzyme proteins with manganese ions as the active center, especially multi-subunit enzymes, such as manganese superoxide dismutase, arginase, manganese catalase, pyruvate carboxylase, oxalate decomposing enzymes (including oxalate oxidase, oxalate decarboxylase and oxalyl-CoA decarboxylase/formyl-CoA transferase, etc.), are almost all multimeric enzymes containing 2-8 identical subunits, wherein manganese superoxide dismutase is 2-mer (prokaryote) or 4-mer (eukaryote), arginase, manganese catalase and pyruvate carboxylase are all 4-subunit, oxalate oxidase is mostly 6-mer, oxalate decarboxylase has 3-mer and 6-mer, oxalyl-CoA decarboxylase is 4-mer, and formyl-CoA transferase is 2-mer. The molecular weight of the polymer is usually 120kDa or more, and usually forms inclusion bodies when expressed in Escherichia coli, and it is not always possible to obtain soluble expressed proteins by using the above methods alone or in combination, and even if soluble, the polymers are not necessarily enzymatically active or have low activity. For these complex proteins, it is not easy to find an expression scheme that is soluble and active in E.coli. The results of experiments using chaperone co-expression to improve soluble expression of a protein of interest are not consistent and, to date, the effect of chaperone co-expression on gene expression appears to be protein specific. It is not clear whether the in vivo levels of chaperones are limited in the case of gene overexpression, and normally protein folding eventually reaches a thermodynamically stable state. Particularly unstable proteins may not fold correctly even in the presence of chaperones. It is now understood that different types of chaperones normally act synergistically. Thus, overexpression of only a single chaperone molecule may be less effective.

Metalloproteins are proteins having metal ions or their metal clusters as a prosthetic group or cofactor. In the overall framework of proteins, nearly half belong to metalloproteins. They are functionally diverse and are involved in various aspects of regulation of homeostasis, electron transport, oxidative stress, gene regulation, signal transduction, and substance/energy metabolism in organisms. The active center of the active enzyme is often provided with metal ions or cofactors, and the correct and sufficient combination of the metal ions of the active center and protein subunits near the active center is an important condition for maintaining the complete structure and activity of the active center. To get something like aOxalate decarboxylase (OXDC) derived from Bacillus subtilis is a hexameric enzyme protein containing manganese ion single subunit, and can catalyze oxalate decarboxylation to generate formic acid and CO₂Each subunit containing 0.86-1.14 manganese ions. The manganese ion being mainly Mn²⁺Bound to a cluster consisting of 3 histidines, capable of recognizing and binding oxalate anions. OxDC has 2 domains and 2 manganese ion binding sites per subunit. When the OxDC is expressed in the escherichia coli, an inclusion body is formed, and the adjustment of the manganese ion concentration in a culture medium and a Buffer in the expression and renaturation processes is important for obtaining the active OxDC. Because a set of manganese ion channel protein system which can completely and accurately regulate the concentration of the intracellular manganese ions exists in the escherichia coli, the intracellular manganese ions still maintain a balance along with the increase of the concentration of the extracellular manganese ions.

The research on the transport, metabolism, homeostatic equilibrium regulation and control of metal ions in cells of a living body and related diseases thereof is a leading hot spot in the research fields of bio-inorganic chemistry, chemical biology, biomedicine and the like. Manganese, known as "cell defense" or "life preserver", plays an important role in living organisms, the content of Manganese ions in the body must be maintained at a proper level, and the absence or excess of Manganese can lead to disease or biological toxicity (Adil Anjem et al. Manganese infection is a key element of the oxygen response to hydrogen peroxide in Molecular Microbiology (2009)72(4), 844-). Therefore, the steady-state balance regulation of the manganese ions in the organism is crucial to the maintenance of the normal physiological functions of the manganese ions in the organism. The manganese homeostasis balance mainly relates to four types of proteins (Liwei, Tantayashi, manganese ion transport, metabolism and homeostatic balance regulation, Life sciences, 2012,24(8): 867-: membrane transporters, chaperonins (chaperoncarperones), manganese storage or utilization proteins (proteins), manganese transcription regulatory proteins (regulatory proteins).

The research of co-expressing molecular chaperones and manganese ion-containing enzyme proteins in escherichia coli, and simultaneously over-expressing or inhibiting manganese ion channel-related protein expression or influencing intracellular manganese ion concentration-related protein to promote soluble and active expression of manganese ion-containing enzyme proteins is not reported in documents at present.

Disclosure of Invention

Aiming at the problems that in the prior art, inactive inclusion bodies are often obtained by expressing recombinant proteins containing manganese ions in escherichia coli, the renaturation process is complex, and the cost is high, the invention aims to provide an escherichia coli expression system of the recombinant proteins containing the manganese ions, the system comprises escherichia coli strains and corresponding plasmids, and soluble and active enzyme proteins containing the manganese ions can be produced by using the system.

The invention also aims to provide a method for efficiently expressing manganese ion-containing enzyme protein by using the escherichia coli expression system.

Compared with the prior art, the technical scheme provided by the invention has the following advantages: (1) most of the manganese ion-containing recombinant protein expressed by the escherichia coli is soluble and active; (2) the separation and purification process of the manganese ion-containing recombinant protein is simple; (3) the total production cost is low.

In order to achieve the purpose, the invention adopts the technical scheme that:

the invention provides an escherichia coli expression system containing manganese ion recombinant protein, wherein a recombinant expression plasmid in the escherichia coli expression system is one or a combination of the following situations:

(1) at least comprises an escherichia coli molecular chaperone gene and an enzyme protein gene containing manganese ions;

(2) comprises escherichia coli molecular chaperone gene, manganese ion-containing enzyme protein gene and overexpression or inhibition manganese ion channel related protein gene, namely overexpression promotes any one or more combinations of manganese ion pumping related channel protein/regulatory protein gene, or knock-out or inhibition or inactivation of manganese ion pumping protein/manganese ion negative feedback inhibition and regulation related protein gene;

(3) comprises an escherichia coli molecular chaperone gene, an enzyme protein gene containing manganese ions and a catalase protein gene.

The manganese ion channel related protein comprises: manganese ions are pumped into the protein MntH, the manganese ions are pumped out of the protein MntP, the manganese ion chaperone protein MntS,manganese transcriptional regulators MntR, OxyR (regulator of the antioxidant system of E.coli) and Fur (with Mn)²⁺Binding inhibition of Fe²⁺Transporter Feo expression), etc.; in addition, intracellular H₂O₂Concentration, superoxide dismutase activity for scavenging oxygen free radicals, peroxidase and catalase activity, intracellular and extracellular Fe²⁺Concentration, etc., all affect the intracellular manganese ion concentration.

The current Escherichia coli expression system often causes the situation that the expressed protein forms inclusion bodies or is degraded by protease, and the biological activity of the expression product is lower. Especially for certain manganese ion-active central multi-subunit enzymes, co-expression of the same chaperone as the source of the target protein may be necessary in some cases for soluble expression of the protein of interest. The invention also finds that the overexpression of related protein genes (such as manganese ion pumping protein genes and the like) for promoting the increase of the intracellular manganese ion concentration or the suppression of related protein genes (such as manganese ion pumping protein and the like) for reducing the intracellular manganese ion concentration is beneficial to improving the soluble expression of the manganese ion-containing recombinant protein.

Preferably, the channel protein gene related to promoting the pumping of manganese ions is MntH protein derived from escherichia coli or homologous protein of MntH protein derived from other species; the regulatory protein gene related to promoting the pumping of the manganese ions is MntS/OxyR protein derived from escherichia coli, or the MntS/OxyR protein is homologous protein derived from other species. In order to improve the soluble and active expression effect of the manganese ion-containing enzyme protein, more preferably, the manganese ion pumping promotion related regulatory protein gene MntS/OxyR protein gene is over-expressed; most preferably, the OxyR protein gene is overexpressed.

Preferably, the manganese ion pump-out protein is a MntP protein derived from escherichia coli or the MntP protein is a homologous protein derived from other species; the manganese ion negative feedback inhibition regulation related protein is manganese ion transcription regulatory protein MntR protein derived from escherichia coli or homologous protein of MntR protein derived from other species. To enhance the soluble and active expression effect of the manganese ion-containing enzyme protein, it is more preferable to knock out/inhibit/inactivate the MntP protein gene.

Preferably, the molecular chaperone gene is any one or more of dnaK-dnaJ-grpE, groES-groEL-tig or tig. More preferably, the chaperone gene is groES-groEL or/and groES-groEL-tig, and most preferably, the chaperone gene is groES-groEL.

Constructing an Escherichia coli strain for expressing molecular Chaperone genes, wherein plasmids for expressing the molecular chaperones can be Chaperone Plasmid Set series plasmids of Takara company, and comprise pG-KJE8, pGro7, pKKE 7, pG-Tf2 and pTf 16; preferably the chaperone plasmid is pG-KJE8, pGro7 or pG-Tf2, most preferably the chaperone plasmid is pGro 7. The original host strain of the recombinant strain of Escherichia coli may be selected from commercially available strains BL21(DE3), BL21trxB (DE3), Rosetta (DE3), Origami2(DE3), Origami B (DE3), Rosetta-gami 2(DE3), Rosetta-gami B (DE3), etc., preferably BL21(DE3) and Origami2(DE3) strains, more preferably Origami2(DE3) strains.

Preferably, the manganese ion-containing enzyme protein gene includes any one or more of an oxalate-lyase gene, an arginase gene, a manganese superoxide dismutase gene, or a manganese catalase gene. More preferably, the enzyme protein gene containing manganese ions is any one or more of oxalate decarboxylase gene A2 shown in sequence SEQ ID NO.1, oxalate decarboxylase gene VL3 shown in sequence SEQ ID NO.2, oxalate oxidase gene B10 shown in sequence SEQ ID NO.3, human arginase gene ARG1 shown in sequence SEQ ID NO.4, or human manganese superoxide dismutase gene hMn-SOD shown in sequence SEQ ID NO. 5. The above genes can be synthesized by whole genes.

Preferably, the same or different medium-strength promoters or weak promoters are adopted as the promoters in front of the molecular chaperone gene/manganese ion channel related protein genes; the promoter sequence is any one of a P43 promoter sequence shown as a sequence SEQ ID NO.6, or an M1-93 promoter shown as a sequence SEQ ID NO.7, or an araBAD promoter shown as a sequence SEQ ID NO.8, or a Lac promoter shown as a sequence SEQ ID NO. 9. The above genes can be synthesized by whole genes.

In order to increase the intracellular manganese ion concentration in the process of culturing the escherichia coli recombinant strain, the conceivable scheme is to increase the manganese ion concentration in the culture medium during culturing the recombinant strain, but because the intracellular manganese ion of the escherichia coli is accurately regulated and controlled by various regulation proteins and ions, the intracellular manganese ion concentration cannot rise along with the increase of the extracellular manganese ion concentration after reaching the equilibrium state, and the method is not effective. In the case of sufficiently high concentrations of manganese ions in the extracellular environment (greater than the steady-state concentrations of manganese ions in the cells), the following technical solutions were studied and obtained:

the invention also provides a method for efficiently expressing the manganese ion-containing enzyme protein by using the escherichia coli expression system, and when the manganese ion-containing enzyme protein is induced and expressed by using the escherichia coli expression system, any one or more of the following treatments are carried out: over-expressing the molecular chaperone gene and manganese ion-containing enzyme protein gene; over-expression to promote the pumping of manganese ion into relevant channel protein/regulatory protein gene; knocking out or inhibiting or inactivating manganese ion pump-out protein/manganese ion negative feedback inhibition regulation related protein genes; over-expression of catalase gene or knock-out of certain gene to reduce intracellular H₂O₂And (4) content.

More preferably, the processing step (ii) specifically includes: a. over-expressing manganese ion pump protein MntH from escherichia coli or homologous protein of other species with similar functions with the protein; b. over-expressing manganese ion chaperonin MntS from escherichia coli or homologous protein of other species with similar functions with the protein; c. over-expressing the OxyR protein from Escherichia coli or homologous proteins of other species having similar functions to the protein;

the processing step three specifically comprises: d. knocking out or inhibiting or inactivating manganese ion pump-out protein MntP from escherichia coli; e. knocking out or inhibiting or inactivating a manganese transcription regulatory protein MntR derived from Escherichia coli.

If a single scheme is adopted in the scheme, a more preferable scheme is to process any one of a to c in the formula II, wherein the more preferable scheme is b or c, and the most preferable scheme is c;

if a combination scheme is adopted in the above scheme, a more preferred scheme is any one of a + b, a + d, b + e and b + c combinations, wherein a + b or b + c combination is more preferred, and a b + c combination is most preferred.

Preferably, when the escherichia coli expression system induces and expresses the manganese ion-containing enzyme protein, the used culture medium is an LB culture medium or a JL culture medium, and the JL culture medium comprises: yeast extract 0.5-1% (w/v), tryptone 1-2% (w/v), KH₂PO₄ 10-25mM，(NH₄)₂SO₄10-50mM, 1-3% (w/v) mannitol, 5-30mM sodium succinate, MgSO₄0.1-0.6mM, initial pH 6.5.

Preferably, when the escherichia coli expression system induces and expresses manganese ion-containing zymoprotein, MnCl is supplemented into the culture medium₂Or MnSO₄To a final concentration of 1-10 mM.

Compared with the prior art, the invention has the beneficial effects that: the invention constructs a new escherichia coli expression system of manganese ion-containing recombinant protein by selecting ideal molecular chaperone protein, optimizing a method for improving manganese ion concentration in escherichia coli cells, coexpression of three proteins, optimizing culture medium composition and other means, provides a production process for expressing manganese ion-containing recombinant protein by various selectable and more optimized escherichia coli engineering bacteria, and provides a possible development direction and a technical route for finally finding out the optimal combination for expressing manganese ion-containing recombinant protein. Compared with the traditional escherichia coli expression system, the expression system developed by the invention improves the activity of the recombinant protein containing manganese ions by more than tens of times, and provides beneficial theoretical and practical basis for large-scale industrial production of the recombinant protein containing manganese ions in escherichia coli.

Drawings

FIG. 1 is a schematic diagram of the design of manganese ion-containing enzyme protein gene recombinant expression plasmid.

FIG. 2 is an oxalate decarboxylase A2 gene expression plasmid pET28a-A2 map.

FIG. 3 is an oxalate decarboxylase A2 gene expression plasmid pET28a-A2-MntH map.

FIG. 4 is a comparison of the expression of oxalate decarboxylase A2 by different strains according to example 5; sample numbers are specified below:

1# pET28a-A2/BL21(DE3) whole liquid

2# pET28a-A2/BL21(DE3) supernatant

3# pET28a-A2-MntH/BL21(DE3) whole liquid

4# pET28a-A2-MntH/BL21(DE3) supernatant

5# (pET28a-A2-MntH pG-KJE8)/BL21(DE3) whole liquid

6# (pET28a-A2-MntH pG-KJE8)/BL21(DE3) supernatant

7# (pET28a-A2-MntH pGro7)/BL21(DE3) whole liquid

8# (pET28a-A2-MntH pGro7)/BL21(DE3) supernatant

9# (pET28a-A2-MntH pKJE7)/BL21(DE3) whole liquid

10# (pET28a-A2-MntH pKJE7)/BL21(DE3) supernatant

11# (pET28a-A2-MntH pG-Tf2)/BL21(DE3) whole liquid

12# (pET28a-A2-MntH pG-Tf2)/BL21(DE3) supernatant

13# (pET28a-A2-MntH pTf16)/BL21(DE3) whole liquid

14# (pET28a-A2-MntH pTf16)/BL21(DE3) supernatant

15# (pET28a-A2pG-KJE8)/BL21(DE3) whole liquid

16# (pET28a-A2pG-KJE8)/BL21(DE3) supernatant

17# (pET28a-A2pGro7)/BL21(DE3) whole liquid

18# (pET28a-A2pGro7)/BL21(DE3) supernatant

19# (pET28a-A2pKJE7)/BL21(DE3) whole liquid

20# (pET28a-A2pKJE7)/BL21(DE3) supernatant

21# (pET28a-A2pG-Tf2)/BL21(DE3) whole liquid

22# (pET28a-A2pG-Tf2)/BL21(DE3) supernatant

23# (pET28a-A2pTf16)/BL21(DE3) whole liquid

24# (pET28a-A2pTf16)/BL21(DE 3).

FIG. 5 is a comparison of the expression of oxalate decarboxylase A2 by different strains according to example 6; sample numbers are specified below:

1# (pET28a-A2-MntH pGro7)/BL21(DE3) whole liquid

2# (pET28a-A2-MntH pGro7)/BL21(DE3) supernatant

3# (pET28a-A2-MntS pGro7)/BL21(DE3) whole liquid

4# (pET28a-A2-MntS pGro7)/BL21(DE3) supernatant

5# (pET28a-A2-OxyR pGro7)/BL21(DE3) whole liquid

6# (pET28a-A2-OxyR pGro7)/BL21(DE3) supernatant

7# pGro7/BL21(DE3) whole liquid

8# pGro7/BL21(DE3) supernatant.

FIG. 6 is a comparison of the expression of oxalate decarboxylase A2 by different strains according to example 7; sample numbers are specified below:

1# pGro7/Origami2(DE3) Whole liquid

2# pGro7/Origami2(DE3) supernatant

3# (pET28a-A2pGro7)/Origami2(DE3) whole liquid

4# (pET28a-A2pGro7)/Origami2(DE3) supernatant

5# (pET28a-A2pGro7)/Origami2(DE3MntP:: BleoR) whole solution

6# (pET28a-A2pGro7)/Origami2(DE3MntP:: BleoR) supernatant

7# (pET28a-A2pGro7)/Origami2(DE3MntR:: BleoR) whole solution

8# (pET28a-A2pGro7)/Origami2(DE3MntR:: BleoR) supernatant.

FIG. 7 is a map of oxalate decarboxylase A2 gene expression plasmid pGEL-MntH-A2.

FIG. 8 is a map of oxalate decarboxylase A2 gene expression plasmid pGEL-MntH-MntS-A2.

FIG. 9 is a comparison of the activities of the supernatant oxalate decarboxylase of the disrupted broth of different strains according to example 9; sample numbers are specified below:

1# pGEL-MntH-A2/Origami2(DE3) supernatant

2# pGEL-MntH-MntS-A2/Origami2(DE3) supernatant

3# pGEL-MntH-A2/BL21(DE3) supernatant

4# pGEL-MntH-MntS-A2/BL21(DE 3).

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the accompanying drawings, and it is to be understood that the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments, and the following embodiments are provided to better illustrate and explain the contents of the present invention. The present invention may be better understood and appreciated by those skilled in the art with reference to the following examples. However, the protection of the invention and the scope of the claims are not limited to the examples provided. 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 experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The invention inserts the enzyme protein gene containing manganese ions into an escherichia coli expression vector, and the escherichia coli expression vector can be pET series, pCold series, pGEX series vectors or other vectors which can be used for expressing protein in an escherichia coli system. In the following examples, pET-28a (+) vector and A2 gene, which are commonly used in pET series vectors, were used as examples to construct recombinant plasmids for expressing manganese ion protein, and other genes were used to test the effect of the E.coli expression system. The amino acid sequence corresponding to the A2 gene is shown as SEQ ID NO.16, the amino acid sequence corresponding to the VL3 gene (derived from enoki mushroom oxalate decarboxylase VEL) is shown as SEQ ID NO.17, the amino acid sequence corresponding to the oxalate oxidase gene B10 is shown as SEQ ID NO.18, the amino acid sequence corresponding to the AGR1 gene is shown as SEQ ID NO.19, and the amino acid sequence corresponding to the hMn-SOD gene is shown as SEQ ID NO. 20.

In the embodiment of the invention, plasmids expressing molecular chaperones and competent cells prepared from Esceichia coli BL21 which respectively converts a chaperonin plasmid are from Takara company, and other Escherichia coli strains and plasmids used in the invention are purchased by companies selling conventional biological materials at home and abroad; the molecular biological reagents used in the present invention were purchased from Thermofish and TOYOBO; the seamless cloning kit was purchased from Biotechnology Inc. of King Nuo Zan, Nanjing (http:// www.vazyme.com /); other common biochemical reagents are all commercially available analytical purifications; the methods of PCR product recovery and gel recovery of DNA were performed using the kit from omega.

In the embodiment, the determination method of the activity of the oxalate oxidase adopts an HPLC detection method, and comprises the following specific steps: mixing 50mM citric acid-NaOH (pH 5.0) and 100mM oxalic acid stock solution in proportion to prepare 0, 0.3, 0.6, 0.9, 1.2, 1.5, 1.8 and 2.1mM oxalic acid standard solutions respectively; adding 10-20 μ L oxalate oxidase-containing solution (protein concentration 0.1-0.2mg/mL) into 1mL 2.5mM oxalic acid solution (pH5.5), reacting at 37 deg.C and 800rpm for 20min, adding 50 μ L2.5M H₂SO₄The reaction was terminated. Sample treatment: firstly, centrifuging a sample after reaction termination at 12000rpm for 10min, and transferring a supernatant into a liquid-phase sample injection bottle; ② centrifuging the sample at 12000rpm for 10min, taking the supernatant, filtering the supernatant into a liquid phase sample bottle by using a 0.45 μm membrane. Detection conditions are as follows: the sample volume is 20 mu L; column temperature: 55 ℃; mobile phase: 2.5mM H₂SO₄(ii) a The flow rate is 0.6 mL/min; a chromatographic column: 0.5 percent of Sepax Carbomix H-NP 10; the sampling time is 22 min. And (4) measuring an oxalic acid standard sample, and obtaining a corresponding integral area drawing standard curve corresponding to the sample with the corresponding concentration through a liquid phase map. And detecting the treated sample by HPLC, processing the obtained data map to obtain the corresponding oxalic acid area, and calculating the enzyme activity unit according to the reduction of oxalic acid. The unit of enzyme activity is defined as the amount of enzyme required to consume 1. mu. mol of oxalic acid or to produce 1. mu. mol of formic acid per minute, and 1 unit of activity.

In the embodiment, the determination of the activity of the arginase is performed by determining the amount of urea released when the arginase catalyzes arginine by adopting a fatty acid reagent method, the arginase is calculated, and the amount of the enzyme which catalyzes and generates 1 mu mol of urea per minute at 37 ℃ is 1 activity unit (Zhengying. cloning and expression of human type I arginase gene, Nanjing university of science and technology, 2006).

In the embodiment hMn, the method for measuring the activity of superoxide dismutase is a method for measuring the activity of superoxide dismutase by adopting improved pyrogallol autoxidation (Dengbuy, Yuanqin, Livingjie. the method for measuring the activity of superoxide dismutase by adopting the improved pyrogallol autoxidation. the biochemical and biophysical progresses, 1991,18(2):89), a BSA kit is adopted for measuring the protein concentration, and the data of specific activity is obtained by dividing the hMn-SOD activity by the protein concentration.

Example 1 design of expression cassette of manganese ion-containing enzyme protein Gene

The schematic diagram of the universal vector for co-expressing the escherichia coli molecular chaperone gene, the manganese ion-containing enzyme protein gene and the manganese ion pumping promotion related channel protein gene constructed in the example is shown in fig. 1. Wherein the promoter 1 and the promoter 2 are medium-strength promoters or weak promoters which can promote the expression of molecular chaperone genes and manganese ion channel related protein genes in Escherichia coli, such as P43 promoter sequence (SEQ ID NO.6, from Bacillus subtilis), M1-93 promoter (SEQ ID NO.7), araBAD promoter (SEQ ID NO.8) and Lac promoter (SEQ ID NO. 9). The promoter 1 and the promoter 2 may be the same promoter, or different promoters may be used. The molecular chaperone gene is one or more of dnaK-dnaJ-grpE, groES-groEL, groES-groEL-tig or tig. The manganese ion channel related protein is one or a combination of more of MntH, MntS, OxyR, catalase protein and the like.

Example 2 construction of oxalate decarboxylase A2 Gene expression plasmid pET28a-A2

A2 gene synthesized by whole gene (SEQ ID NO.1) is used as a template, a primer pair F1/R1 is designed, the gene is amplified, and an amplification product is recovered and purified by glue, and the method refers to a method of a commercial DNA small purification kit instruction, so that a DNA fragment 1 (namely an A2 gene fragment) is finally obtained. The PCR system is as follows: 10 XPCR Buffer 5. mu.L, 2mM dNTP 5. mu.L, 25mM MgSO₄mu.L of 5. mu.L, 1.5. mu.L each of 10. mu.M primer F/R, 0.5. mu.L of template DNA, KOD-Plus-Neo 1. mu.L, ddH2O 32.5.5. mu.L; the PCR reaction conditions were as follows: 3min at 94 ℃,30 cycles (98 ℃ for 10s, 60 ℃ for 30s, 68 ℃ for 35s), 5min at 68 ℃ and 10min at 4 ℃; the PCR system in the following description of vector construction is identical to the above description, and will not be described in detail below, and the PCR reaction conditions are slightly different, mainly the annealing temperature and the extension time. Using commercially available pET-28a plasmid as template, designing primer pair F2/R2, amplifying plasmid, annealing at 55 deg.C for 5min, and digesting the amplified product with restriction enzyme Dpn I at 37 deg.C for 2h (50 μ L system, reference reaction conditions for reference) as the PCR conditions for A2 gene amplificationInstruction book), and performing gel recovery and purification on the digested amplification product to finally obtain a DNA fragment 2 (namely pET-28a fragment). The reaction system shown in the following table was prepared in an ice water bath by the method of a seamless cloning kit using the method of the seamless cloning kit instructions, and the above DNA fragment 1 and DNA fragment 2 were ligated to transform E.coli DH5 α.

ddH2O	Up to 20μl
		5xBuffer	4μl
DNA fragment 2 (i.e., pET-28a fragment)	80ng
		DNA fragment 1 (i.e., A2 gene fragment)	50ng
Recombinant enzyme	2μl

DH 5. alpha. super-competence was prepared by Inoue method, referred to molecular cloning, instruction (3 rd edition), spread on resistant LB solid medium plates containing 50. mu.g/ml kanamycin for screening, positive clones were verified by PCR and sequencing, and the correctly sequenced recombinant plasmid was named pET28a-A2 (FIG. 2); the sequences of the primers used above are as follows:

F1：5’-AGAAGGAGATATACCATGGCTCCAGCACCTTCCAG-3’

R1：5’-GTTAGCAGCCGGATCCTAAGCAGGACCGACCACAAT-3’

F2：5’-GATCCGGCTGCTAACAAAGC-3’

R2：5’-GGTATATCTCCTTCTTAAAG-3’

example 3 chaperone overexpression Strain construction

The 5 commercial chaperone plasmids pG-KJE8, pGro7, pKJE7, pG-Tf2 and pTf16 were transformed into commercial strains, positive clones were selected on LB solid medium plates containing 20. mu.g/ml, and construction of chaperone overexpression strains was carried out using BL21(DE3) and Origami2(DE3) expression strains as host bacteria. Competent cell preparation and E.coli transformation were referred to in molecular cloning guidelines (3 rd edition) to obtain a series of strains: pG-KJE8/BL21(DE3), pGro7/BL21(DE3), pKJE7/BL21(DE3), pG-Tf2/BL21(DE3), pTf16/BL21(DE3), pG-KJE8/Origami2(DE3), pGro7/Origami2(DE3), pKJE7/Origami2(DE3), pG-Tf2/Origami2(DE3) and pG pTf16/Origami2(DE 3).

Example 4 construction of Co-expression vector for protein A2 and manganese ion channel-related protein

Using the synthesized P43 promoter DNA (SEQ ID NO.6) fragment as a template, designing a primer pair F3/R3 for amplification, and purifying a product to obtain a DNA fragment 3; extracting the genome DNA of Escherichia coli JM109 by using a bacterial genome DNA extraction kit, designing a primer pair F4/R4 to amplify MntH gene (SEQ ID NO.10) by using the genome DNA of Escherichia coli K12MG1655 strain as a template, and purifying the amplified product by using a DNA purification kit to obtain a DNA fragment 4; using a synthesized terminator (SEQ ID NO.11) as a template, designing a primer pair F5/R5 for amplification, and purifying a product to obtain a DNA fragment 5; using plasmid pET28a-A2 as a template, designing a primer F6/R6 for amplification, and obtaining a DNA fragment 6 after purifying a product; the above DNA fragments 3, 4, 5 and 6 were ligated by means of a seamless cloning kit, E.coli DH 5. alpha. was transformed, plated on resistant LB solid medium plates containing 50. mu.g/ml kanamycin for selection, positive clones were verified by PCR and sequencing, and the sequencing-correct vector was named pET28a-A2-MntH (FIG. 3). The PCR amplification and seamless cloning system and method used in this example are similar to those of example 2 and are not described in detail herein. The sequences of the primers used above are as follows:

F3：5’-GGATCTCAACTCGAGTGATAGGTGGTATGTTTTCG-3’

R3：5’-GCGATAGTTCGTCATCGTTCATGTCTCCTTTTTTA-3’

F4：5’-AAGGAGACATGAACGATGACGAACTATCGCGTTGA-3’

R4：5’-TAGTGCTCGAATTCATTACAATCCCAGTGCCGTAC-3’

F5：5’-GCACTGGGATTGTAATGAATTCGAGCACTAGTGCA-3’

R5：5’-AGGATCTTCAAGCTTGGAAGGAAATGATGACCTCG-3’

F6：5’-AAGCTTGAAGATCCTTTGATC-3’

R6：5’-CTCGAGTTGAGATCCTTTTTTTC-3’

taking genome DNA of Escherichia coli K12MG1655 strain as a template, designing a primer pair F7/R7 to amplify MntS gene (SEQ ID NO.12), and purifying the amplified product by a DNA purification kit method to obtain a DNA fragment 7; using pET28a-A2-MntH plasmid as a template, designing a primer pair F8/R8 for amplification, and purifying a product to obtain a DNA fragment 8; the above DNA fragments 7 and 8 were ligated by means of a seamless cloning kit, E.coli DH 5. alpha. was transformed, plated on resistant LB solid medium plates containing 50. mu.g/ml kanamycin for screening, positive clones were verified by PCR and sequencing, and the correctly sequenced vector was named pET28 a-A2-MntS. The sequences of the primers used above are as follows:

F7：5’-AAGGAGACATGAACGATGAATGAGTTCAAGAGGTG-3’

R7：5’-TAGTGCTCGAATTCATTATTTATCGGAAGGTTTAT-3’

F8：5’-TAATGAATTCGAGCACTAGTGCAGC-3’

R8：5’-CATCGTTCATGTCTCCTTTTTTATG-3’

taking the genome DNA of the Escherichia coli K12MG1655 strain as a template, designing a primer pair F9/R9 to amplify an OxyR gene (SEQ ID NO.13), and purifying the amplified product by a DNA purification kit method to obtain a DNA fragment 9; using pET28a-A2-MntH plasmid as a template, designing a primer pair F8/R8 for amplification, and purifying an amplification product to obtain a DNA fragment 10; the above DNA fragments 9 and 10 were ligated by means of a seamless cloning kit, E.coli DH 5. alpha. was transformed, plated on resistant LB solid medium plates containing 50. mu.g/ml kanamycin for screening, positive clones were verified by PCR and sequencing, and the vector with the correct sequencing was named pET28 a-A2-OxyR. The sequences of the primers used above are as follows:

F9：5’-AAGGAGACATGAACGATGAATATTCGTGATCTTGAG-3’

R9：5’-TAGTGCTCGAATTCATTAAACCGCCTGTTTTAAAAC-3’

example 5A2 protein, chaperone protein and MntH protein Co-expression assay

Plasmid DNAs pET28a-A2 and pET28a-A2-MntH were extracted according to the method of a plasmid minilab kit, respectively, the Escherichia coli BL21(DE3) strain was transformed, spread on a resistant LB solid medium plate containing 50. mu.g/ml kanamycin and screened, and positive clones were verified by PCR to obtain recombinant strains pET28a-A2/BL21(DE3) and pET28a-A2-MntH/BL21(DE 3). pET28a-A2 and pET28a-A2-MntH plasmids were transformed into molecular chaperone overexpression strains pG-KJE8/BL21(DE3), pGro7/BL21(DE3), pKJE7/BL21(DE 21), pG-Tf 21/BL 21(DE 21) and 21/BL 21(DE 21), respectively, plated on resistant LB solid medium plates containing 20. mu.g/ml chloramphenicol and 50. mu.g/ml kanamycin, positive clones were screened by PCR to obtain recombinant strains (pET 28-21-MntH pG3672)/BL 21(DE 21), (pET28 21-A21-MntH-21)/BL 21/pET 21(DE 21), (pGT 21-MntH-21)/pET 21-21 (DE 21-MntH 21)/pET 21/21) by PCR, (pET28a-A2pG-KJE8)/BL21(DE3), (pET28a-A2pGro7)/BL21(DE3), (pET28a-A2 pKKE 7)/BL21(DE3), (pET28a-A2pG-Tf2)/BL21(DE3) and (pET28a-A2pTf16)/BL21(DE 3).

The monoclonal antibodies on the resistant plates were inoculated into LB liquid seed medium (yeast extract 0.5% (w/v), tryptone 1% (w/v), NaCl 1% (w/v), 20. mu.g/ml chloramphenicol, 50. mu.g/ml kanamycin) and cultured until OD600 became 1.0-1.2, and into JL medium (yeast extract 0.75% (w/v), tryptone 1.5% (w/v), KH) at an inoculum size of 2% (v/v)₂PO₄ 15mM，(NH₄)₂SO₄25mM, mannitol 1.8% (w/v), sodium succinate 20mM, MgSO₄0.25mM, initial pH 6.5.) at 37 deg.C, and shaking table rotation speed of 150 rpm, culturing until OD600 reaches 1.0, and adding inducer, wherein all strains need to be supplementedAdding IPTG to a final concentration of 0.5mM and MnCl to a final concentration of 5mM₂Or MnSO₄The bacterial strain expressing the molecular chaperone needs to be additionally supplemented with L-arabinose 0.6mg/mL or 5ng/mL tetracycline or both (refer to the specification of molecular chaperone plasmid specifically), the temperature is reduced to 28 ℃ during induction, the culture is stopped until OD600 is about 8.0, 13000g of 1.5mL bacterial liquid is taken for centrifugation for 5min, the supernatant is removed, 1.5mL of 0.9% physiological saline is added for cleaning the bacterial strain once, 13000g is centrifuged for 5min, the supernatant is removed, 1.5mL of 50mM Tris-HCl (8.0) Buffer is added, and the bacterial strain is subjected to ultrasonic lysis after being fully suspended. And (4) taking the cell lysate whole liquid and detecting the activity of the supernatant enzyme. The oxalate decarboxylase enzyme activity was measured by HPLC (M Kesarwani et al, the Journal of Biological Chemistry,2000,275: 7230-7238). As a result of shake flask fermentation, the strains in which the A2 gene alone was expressed and the A2 gene was co-expressed with MntH were found to have no oxalate decarboxylase activity in either the whole cell disruption solution or the supernatant of the cell disruption solution (FIG. 4); the plasmid co-expressed by the A2 gene and MntH is transferred into bacterial strains pG-KJE8/BL21(DE3), pGro7/BL21(DE3) and pG-Tf2/BL21(DE3) over-expressing molecular chaperones, so that the activity can be detected in the whole liquid and the supernatant of cell disruption liquid; the A2 gene and molecular chaperone coexpressed strain, similarly, only the cell disruption whole liquid and supernatant of the strain containing pG-KJE8, pGro7 and pG-Tf2 had weak activity. As can be seen from the enzyme activity data, insoluble protein is hardly active, and the expression effect of the molecular chaperone plasmid pGro7 is the best, so the molecular chaperone gene of the invention is preferably groES-groEL from pGro7 plasmid, that is, the overexpression of MntH and the proper expression of the groES-groEL molecular chaperone in Escherichia coli help oxalate decarboxylase to form soluble and active protein.

Example 6 Effect of MntH, MntS and OxyR overexpression on A2 protein expression

Plasmid DNAs pET28a-A2-MntH, pET28a-A2-MntS and pET28a-A2-OxyR obtained in example 4 were extracted according to a plasmid mini-kit method, and molecular chaperone expression strains pGro7/BL21(DE3) were transformed, spread on resistant LB solid medium plates containing 50. mu.g/ml kanamycin and 20. mu.g/ml chloramphenicol for screening, and positive clones were verified by PCR to obtain recombinant strains (pET28a-A2-MntH pGro7)/BL21(DE3), (pET28a-A2-MntS pGro7)/BL21(DE3) and (pET 28-A2-OxyR pG 7)/BL21(DE 686 3). The same medium and expression conditions as those used in example 5 were used for expression, and pGro7/BL21(DE3) strain was used as a control, and the results of enzyme activities are shown in FIG. 5. As shown in FIG. 5, it is understood that the overexpression of OxyR has the best effect of promoting the soluble and active expression of A2 protein, and MntS is the order of magnitude.

Example 7 inactivation of the MntP and MntR genes

The method for knocking out genes in escherichia coli is multiple, the simplest insertion inactivation method is selected in the embodiment, the fragments of 150bp at the upstream and downstream of MntP and MntR genes are fused with the resistance genes to obtain fusion fragments, and the transformed escherichia coli can be screened by a resistance plate to screen inactivated positive clones of the MntP and MntR genes. Conveniently, a fusion DNA fragment (MntPOP 100-BleoR-MntPown 100) of three DNA fragments, namely an upstream 100bp fragment (MntpUP100) of the MntP gene, a Zeocin resistance gene (BleoR) from a Pichia pastoris expression vector pPICZ alpha A and a downstream 100bp fragment (MntpDOwn100) of the MntP gene, is synthesized by a whole-gene synthesis method, and a fusion DNA fragment (MntRUP 100-BleoR-MntDOwn 100) is synthesized by the same method. The fusion fragments MntPOP 100-BleoR-MntPown 100 (shown by sequence SEQ ID NO. 14) and MntROR 100-BleoR-MntROwn 100 (shown by sequence SEQ ID NO. 15) were transformed into pGro7/Origami2(DE3) respectively, applied to solid LB medium (yeast extract 0.5% (w/v), peptone 1% (w/v), NaCl 0.5% ((w/v), Zeocin concentration 25. mu.g/ml, agarose 2%, pH7.5) plates, positive strains were selected, and the resulting positive strains were named pGro7/Origami2(DE3MntR:: BleoR) and pGrO7/Origami2(DE3 MntROR:: BleoR) by PCR and sequencing verification.

The primers for verifying the MntP gene are as follows:

F10：5’-TGCATCAATCGGTAAAGGTG-3’

R10：5’-GAAGTGCGTCCAGAGGATCT-3’

verifying that the MntR gene primer is as follows:

F11：5’-AAGTGACGCAGTTAGTGAACG-3’

R11：5’-CACCGTGTTTCTGGGTAAAC-3’

pET28a-A2 plasmid DNA was transformed into pGro7/Origami2(DE3), pGro7/Origami2(DE3MntP:: BleoR) and pGro7/Origami2(DE3MntR:: BleoR) strains according to the method of a plasmid minikit, spread on resistant LB solid medium plates containing 50. mu.g/ml kanamycin, screened, and positive clones were verified by PCR to give recombinant strains (pET28a-A2pGro7)/Origami2(DE3), (pET28a-A2pGro7)/Origami2(DE3MntP:: BleoR) and (pET28a-A2pGro7)/Origami2(DE3 eMnoR) strains. The same medium and expression conditions as those used in example 5 were used for expression, and pGro7/Origami2(DE3) strain was used as a control, and the results of enzyme activities are shown in FIG. 6. The inactivation of the MntP gene and the MntR gene is beneficial to improving the expression activity of the A2 protein in escherichia coli, and the expression activity of the MntP gene is better than that of the MntR gene when the MntP gene is inactivated, but the effect is worse than that of the over-expression of MntH, MntS and OxyR on the A2 protein.

Example 8 pGEL-MntH-A2 and pGEL-MntH-MntS-A2 vector construction

In order to further optimize the expression of A2 protein in Escherichia coli, improve the stability of recombinant strains, reduce the loss rate of plasmids and promote the expression of A2, molecular chaperone genes, A2 genes and manganese ion channel related protein (MntH, MntS, OxyR and the like) genes are constructed on the same carrier, and the influence of different manganese ion channel protein combinations on the expression of A2 is tried.

Using pGro7 plasmid DNA as a template, designing a primer pair F12/R12 to amplify a molecular chaperone gene cluster groES-groEL, and purifying an amplified product by a DNA purification kit to obtain a DNA fragment 11; using pET28a-A2-MntH plasmid as a template, amplifying by using a primer F8/R8, and purifying a product to obtain a DNA fragment 12; the above DNA fragments 11 and 12 were ligated by means of a seamless cloning kit, E.coli DH 5. alpha. was transformed, plated on resistant LB solid medium plates containing 50. mu.g/ml kanamycin, screened, positive clones were verified by PCR, and the correctly sequenced vector was named pGEL-A2. Using a synthesized M1-93 promoter sequence (SEQ ID NO.7) as a template, designing a primer pair F13/R13 for amplification, and purifying an amplification product by using a DNA purification kit to obtain a DNA fragment 13; pET28a-A2-MntH plasmid is used as a template, a primer pair F14/R14 is designed to amplify DNA fragment containing MntH and a terminator, and the product is purified to obtain DNA fragment 14; taking pGEL-A2 plasmid DNA as a template, designing a primer pair F15/R15 amplification linearized vector, and purifying a product to obtain a DNA fragment 15; the above DNA fragments 13, 14 and 15 were ligated by means of a seamless cloning kit, E.coli DH 5. alpha. was transformed, plated on resistant LB solid medium plates containing 50. mu.g/ml kanamycin for selection, positive clones were verified by PCR and sequencing, and the correctly sequenced vector was named pGEL-MntH-A2 (FIG. 7).

Using pET28a-A2-MntS plasmid as a template, amplifying by using a primer F16/R16, and purifying a product to obtain a DNA fragment 16; taking pGEL-MntH-A2 plasmid DNA as a template, designing a primer pair F17/R17 amplification linearized vector, and purifying a product to obtain a DNA fragment 17; the above DNA fragments 16 and 17 were ligated by means of a seamless cloning kit, E.coli DH 5. alpha. was transformed, plated on resistant LB solid medium plates containing 50. mu.g/ml kanamycin for screening, positive clones were verified by PCR and sequencing, and the correctly sequenced vector was named pGEL-MntH-MntS-A2 (FIG. 8).

The sequences of the primers used above are as follows:

F12：5’-AAGGAGACATGAACGATGAATATTCGTCCATTGC-3’

R12：5’-TAGTGCTCGAATTCATTACATCATGCCGCCCATG-3’

F13：5’-GGATTGGCGAATGGGTTATCTCTGGCGGTGTTGAC-3’

R13：5’-GCGATAGTTCGTCATATGAGCTGTTTCCTGGTTTAAAC-3’

F14：5’-CAGGAAACAGCTCATATGACGAACTATCGCGTTGAG-3’

R14：5’-GAAAAGTGCCACCTGGGAAGGAAATGATGACCTCG-3’

F15：5’-CAGGTGGCACTTTTCGGGGAAATG-3’

R15：5’-CCCATTCGCCAATCCGGATATAG-3’

F16：

5’-GCACTGGGATTGTAGTAAGAAGGAGATATACATATGAATGAGTTCAAG-3’

R16：5’-TAGTGCTCGAATTCACTATTTATCGGAAGGTTTATCTT-3’

F17：5’-TGAATTCGAGCACTAGTGC-3’

R17：5’-CTACAATCCCAGTGCCGTAC-3’

example 9 expression testing of recombinant expression plasmids pGEL-MntH-A2 and pGEL-MntH-MntS-A2

pGEL-MntH-A2 and pGEL-MntH-MntS-A2 plasmid DNAs were extracted according to the plasmid mini-kit method, Origami2(DE3) and BL21(DE3) strains were transformed, respectively, spread on a resistant LB solid medium plate containing 50. mu.g/ml kanamycin and screened, positive clones were verified by PCR to obtain recombinant strains pGEL-MntH-A2/Origami2(DE3), pGEL-MntH-MntS-A2/Origami2(DE3), pGEL-MntH-A2/BL21(DE3) and pGEL-MntH-MntS-A2/BL21(DE3), expressed using the same medium and expression conditions as in example 5, pGro7/Origami2(DE3) as a strain, and the results of enzyme activity were shown in FIG. 9 as a control strain, as a recombinant strain, as shown in FIG. 679

The oxalate decarboxylase activity of the supernatant of the disruption solution of pGEL-MntH-MntS-A2/Origami2(DE3) is the highest, and the Origami2(DE3) strain is better than BL21(DE3) strain during recombination.

Example 10 expression of oxalate decarboxylase VL3 in E.coli

The VL3 gene (SEQ ID NO.2) synthesized by the whole gene is used as a template, a primer pair F18/R18 is designed, the gene is amplified, and an amplification product is recovered and purified by glue, wherein the method refers to a method of a commercial DNA small purification kit instruction, and finally the DNA fragment 18 (namely the VL3 gene fragment) is obtained. The pGEL-MntH-A2 plasmid constructed in example 8 was used as a template, a primer pair F19/R19 was designed, the plasmid was amplified under PCR conditions of 55 ℃ annealing temperature and 5min extension time, and other conditions were the same as those of the PCR conditions for amplification of the A2 gene described in example 2, and the amplification product was digested with restriction enzyme Dpn I at 37 ℃ for 2h (50. mu.L system, reaction conditions refer to the manual), and the gel recovery and purification were performed on the amplification product after digestion, to obtain a DNA fragment 19. A reaction system is prepared in an ice-water bath by a method of a seamless cloning kit and a method of a seamless cloning kit specification, the DNA fragment 18 and the DNA fragment 19 are connected, escherichia coli DH5 alpha is transformed, the escherichia coli DH5 alpha is coated on a resistant LB solid culture medium plate containing 50 mu g/ml kanamycin for screening, a positive clone is verified by PCR and sequencing, and a vector with correct sequencing is named as pGEL-MntH-VL 3.

The sequences of the primers used above are as follows:

F18：5’-AGAAGGAGATATACCATGAACGAAACTTCCGATCCA-3’

R18：5’-GTTAGCAGCCGGATCCTATTAGTTAACTGGACCAAC-3’

F19：5’-GGTATATCTCCTTCTTAAAG-3’

R19：5’-GGTATATCTCCTTCTTAAAG-3’

pET28a-VL3 vector was constructed using the similar method as in example 2. pGEL-MntH-VL3 and pET28a-VL3 vectors were transformed into Origami2(DE3) strain, spread on resistant LB solid medium plate containing 50. mu.g/ml kanamycin to screen, positive clones were verified by PCR to obtain recombinant strains pGEL-MntH-VL3/Origami2(DE3) and pET28a-VL3/Origami2(DE3), and expression was performed using the same medium and expression conditions as in example 5, as a result, it was found that oxalate decarboxylase activity in the supernatant and the whole liquid of the recombinant strain pGEL-MntH-VL3/Origami2(DE3) were 4,820U/L and 5,120U/L, respectively, while oxalate decarboxylase activity was not detected in the supernatant and the whole liquid of the recombinant strain pET28a-VL3/Origami2(DE 3).

Example 11 expression of oxalate oxidase B10 in E.coli overexpressing manganese ion channel protein and chaperone protein

A B10 gene (SEQ ID NO.3) synthesized by a whole gene is used as a template, a primer pair F20/R20 is designed, the gene is amplified, and an amplification product is recovered and purified by glue, wherein the method refers to a method of a commercial DNA small purification kit instruction, and finally a DNA fragment 20 (namely a B10 gene fragment) is obtained. Using pGEL-MntH-A2 plasmid constructed in example 8 as a template, a primer pair F19/R19 was designed, the plasmid was amplified, the amplified product was digested with restriction enzyme Dpn I at 37 ℃ for 2 hours (50. mu.L system, reaction conditions refer to the manual), the reaction conditions refer to the manual, and the digested amplified product was recovered and purified by gel to obtain DNA fragment 21. A reaction system is prepared in an ice-water bath by a method of a seamless cloning kit and a method of a seamless cloning kit specification, the DNA fragment 20 and the DNA fragment 21 are connected, escherichia coli DH5 alpha is transformed, the escherichia coli DH5 alpha is coated on a resistant LB solid culture medium plate containing 50 mu g/ml kanamycin for screening, a positive clone is verified by PCR and sequencing, and a vector with correct sequencing is named as pGEL-MntH-B10.

The primer sequences used were as follows:

F20：5’-AGAAGGAGATATACCATGTCTGATCCTGGTCTCCT-3’

R20：5’-GTTAGCAGCCGGATCTTAAGCAACATCAGTTAAGAG-3’

pET28a-B10 vector was constructed using the similar method as in example 2. pGEL-MntH-B10 and pET28a-B10 vectors were transformed into Origami2(DE3) strain, pET28a-B10 vectors were transformed into pGro7/Origami2(DE3), plated on a resistant LB solid medium plate containing 50. mu.g/ml kanamycin for screening, and positive clones were verified by PCR to give recombinant strains pGEL-MntH-B10/Origami2(DE3), pET28a-B10/Origami2(DE3) and (pET28a-B10pGro7)/Origami2(DE 3). Expression was carried out using the same medium and expression conditions as in example 5, and as a result, it was found that oxalate oxidase activities in the supernatant and the whole liquid of recombinant strains pGEL-MntH-B10/Origami2(DE3) were 46.5U/mL and 47.1U/mL, respectively, whereas oxalate oxidase activities were not detected in both the supernatant and the whole liquid of recombinant strains pET28a-B10/Origami2(DE3) and (pET28a-B10pGro7)/Origami2(DE 3).

Example 12 expression of arginase in E.coli overexpressing manganese ion channel protein and chaperone protein

A primer pair F21/R21 is designed by taking an ARG1 gene (SEQ ID NO.4) synthesized by a whole gene as a template, the gene is amplified, and amplification products are recovered and purified by glue, the method refers to the method of a commercial DNA small purification kit instruction, and finally obtains a DNA fragment 22, the pGEL-MntH-MntS-A2 plasmid constructed in the example 8 is taken as the template, the primer pair F19/R19 is designed, the plasmid is amplified, the amplification products are digested for 2h at 37 ℃ by using restriction enzyme Dpn I (50 muL system, the reaction conditions refer to the instruction), and the digested amplification products are recovered and purified by glue, and finally the DNA fragment 23 is obtained. A reaction system is prepared in an ice-water bath by a method of a seamless cloning kit and a method of a seamless cloning kit specification, the DNA fragment 22 and the DNA fragment 23 are connected, escherichia coli DH5 alpha is transformed, the escherichia coli DH5 alpha is coated on a resistant LB solid culture medium plate containing 50 mu g/ml kanamycin for screening, a positive clone is verified by PCR (polymerase chain reaction) and sequencing, and a vector with correct sequencing is named as pGEL-MntH-MntS-AGR 1.

The sequences of the primers used above are as follows:

F21：5’-AGAAGGAGATATACCATGAGCGCCAAGTCCAGAAC-3’

R21：5’-GTTAGCAGCCGGATCTTACTTAGGTGGGTTAAGGT-3’

the pET28a-AGR1 vector was constructed in a similar manner as in example 2. pGEL-MntH-MntS-AGR1 and pET28a-AGR1 vectors were transformed into Origami2(DE3) strain, pET28a-AGR1 vectors were transformed into pGro7/Origami2(DE3), plated on resistant LB solid medium plate containing 50. mu.g/ml kanamycin for selection, the positive clones were verified by PCR to give recombinant strains pGEL-MntH-MntS-AGR1/Origami2(DE3), pET28a-AGR1/Origami2(DE3) and (pET28a-AGR1pGro7)/Origami2(DE3) which were expressed using the same medium and expression conditions as in example 5, and as a result, it was found that the arginase activities of the supernatants of the recombinant strains pGEL-MntH-MntS-AGR1/Origami2(DE3) were 16.53U/mL, respectively, and the arginase activities of the supernatants of the recombinant strains pET28a-AGR1/Origami2(DE3) and (pET28a-AGR1pGro7)/Origami2(DE3) were 4.16U/mL and 6.54U/mL, respectively.

Example 13 expression of superoxide dismutase in E.coli overexpressing manganese ion channel protein and chaperone protein

A primer pair F21/R21 is designed by taking hMn-SOD gene (SEQ ID NO.5) synthesized by whole gene as a template, the gene is amplified, and amplification products are recovered and purified by glue, the method refers to the method of a commercial DNA small purification kit instruction, and finally a DNA fragment 24 is obtained, the pGEL-MntH-MntS-A2 plasmid constructed in the example 8 is taken as the template, the primer pair F19/R19 is designed, the plasmid is amplified, the amplification products are digested for 2h at 37 ℃ by using restriction enzyme Dpn I (50 muL system, the reaction conditions refer to the instruction), and the digested amplification products are recovered and purified by glue, and finally the DNA fragment 25 is obtained. A reaction system is prepared in an ice-water bath by a method of a seamless cloning kit and a method of a seamless cloning kit specification, the DNA fragment 24 and the DNA fragment 25 are connected, escherichia coli DH5 alpha is transformed, the escherichia coli DH5 alpha is coated on a resistant LB solid culture medium plate containing 50 mu g/ml kanamycin for screening, a positive clone is verified by PCR (polymerase chain reaction) and sequencing, and a vector with correct sequencing is named as pGEL-MntH-MntS-hMn-SOD.

The sequences of the primers used above are as follows:

F22：5’-AGAAGGAGATATACCATGCAGCTGCACCACAGCAAG-3’

R22：5’-GTTAGCAGCCGGATCTTACTTTTTGCAAGCCATGT-3’

pET28a-hMn-SOD vector was constructed in a similar manner to that described in example 2. pGEL-MntH-MntS- -SOD and pET 28- -SOD vector-transformed Origami (DE) strain, pET 28- -SOD vector-transformed pGro/Origami (DE), were spread on a resistant LB solid medium plate containing 50. mu.g/ml kanamycin and screened, positive clones were verified by PCR to obtain recombinant strains pGEL-MntH-MntS- -SOD/Origami (DE), pET 28- -SOD/Origami (DE) and (pET 28- -SOD ro)/Origami (DE) were expressed using the same medium and expression conditions as in example 5, and as a result, it was found that the supernatant-SOD specific enzyme activity of the recombinant strains pGEL-MntH-MntS- -SOD/Origami (DE) was 1254U/mg, while the recombinant strains pET 28- -SOD/Origami (DE) and (pGT 28- -pGro)/Origami (DE) The specific enzyme activity of the supernatant hMn-SOD is 358U/mg and 629U/mg, and the over-expression of MntH and MntS leads the specific activity of hMn-SOD to be improved by 2 times.

The invention finally finds out the optimized combination by screening and testing the overexpression or knockout of different molecular chaperones, different host bacteria and different manganese ion channel related proteins, realizes the high-efficiency expression of target proteins, obtains soluble and active enzymes, has high efficiency compared with the traditional expression mode, simple purification process and low cost, and is beneficial to the industrial production and application of the enzymes.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Sequence listing

<110> Wuhan Kangfu Biotechnology GmbH

<120> Escherichia coli expression system of manganese ion-containing recombinant protein and application method thereof

<160> 20

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1359

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atggctccag caccttccag cgcagcttcc tccatcgttg tctccgctac ttcatcatcg 60

actgtttcga gtgcacccgt gagcgtttcg agcttcctgc ccactacctc cattgctgct 120

gcaactccta gttcaatcgc tgtggcttta tcatccacag ctacggttcc cttcatcgac 180

ttgaatccta atggacctct gtgggacccg tctgtgagcg gtgtacctca ggctgaacgt 240

ggttccttgg gagcaactat catgggacct acagatgtgg acacgacaaa ggcaaatcca 300

gacctgttgg caccacctac tactgaccac ggttctgtag ataatgcaaa atgggcattt 360

tccttatccc ataacagatt gcaaactggc ggttgggcca gggagcaaaa cataggtgcc 420

atgccaattg caactgaaat ggcatctgtc aacatgaggc ttgaaccagg tgctattaga 480

gaattgcact ggcataagac agcagagtgg gcttacgtcc taaagggaaa tactcaggtg 540

actgctgttg atcaaaatgg taaaaacttt atcggtactg taggaccagg tgatctttgg 600

tacttcccac cgggtattcc tcattcgcta caagctacag gtgatgaccc agaaggctca 660

gagttcatac tggttttcga ttctggtgct ttttctgagg attccacctt tttgttgact 720

gattggatga gtcatgttcc agtggaagtc ttggccaaaa acttccagac cgatatctca 780

gcatttgcca gaatcccagc tgaggagttg tatatctttc ccgctgccgt tccacctgat 840

tctcaacaag accctacatc tcctgaagga accgtcccaa atccttttac ttttgcttta 900

tccaaggtcc cacctatgca attgtctgga ggtaccgcaa aaatcgttga ctcaacaact 960

tttaccgttt ctaaggccat cgcagctgcc gaggtaacta tagaaccagg cgctatcaga 1020

gaacttcatt ggcaccccac acaagacgag tggtcatttt tcatcgaggg tagagctaga 1080

atgacaattt tcgccgctca gtctaatgct cgtacattcg actaccaagc cggtgacatt 1140

ggttacgttc ccgcaactat gggacattat gtggagaata ttggaaacac aacagtgcgt 1200

tatctggaga ttttcaacac ggctgttttt gaagatattt ccctcagtaa ttggttagcc 1260

ttaacgccac cagaattggt taaagcacac ttgggtttcg atgacgctac aatggctcac 1320

ttggctaagg taaaaccaat tgtggtcggt cctgcttag 1359

<210> 2

<211> 1176

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atgaacgaaa cttccgatcc agctttggtt aagccagaga gaaaccagtt gggagctact 60

attcaaggtc cagacaactt gccaatcgac ttgcaaaacc cagatttgtt ggctccacca 120

actactgatc acggtttcgt tggtaacgct aagtggccat tctcattctc caagcagaga 180

ttgcaaactg gtggatgggc tagacaacag aacgaagttg ttttgccatt ggctacaaac 240

ttggcttgta caaacatgag attggaggct ggtgctatta gagaattgca ctggcacaag 300

aacgctgaat gggcttacgt tttgaagggt tccactcaga tttctgctgt tgacaacgag 360

ggtagaaact acatctccac tgttggtcct ggtgatttgt ggtacttccc accaggtatt 420

ccacattcct tgcaggctac tgctgatgat ccagaaggtt ccgagttcat cttggttttc 480

gactccggtg ctttcaacga tgacggtact ttcttgttga ctgattggtt gtcccacgtt 540

ccaatggaag ttatcttgaa gaacttcaga gctaagaacc ctgctgcttg gtcacatatt 600

ccagctcaac agttgtacat cttcccatct gaacctccag ctgataacca accagaccca 660

gtttctccac aaggtactgt tccattgcca tactcattca acttctcatc cgttgagcca 720

actcaatact ctggtggtac tgctaagatt gctgactcca ctactttcaa catctccgtt 780

gctattgctg ttgctgaggt tacagttgaa cctggtgctt tgagagagtt gcattggcat 840

ccaactgaag atgagtggac tttcttcatc tccggtaacg ctagagttac tatcttcgct 900

gctcaatccg ttgcttccac tttcgattac cagggtggtg acattgctta cgttccagct 960

tctatgggac actacgttga gaacatcggt aacactactt tgacttactt ggaggttttc 1020

aacactgaca gattcgctga cgtttctttg tctcagtggc ttgctttgac tccaccatct 1080

gttgttcagg ctcacttgaa cttggacgac gaaactttgg ctgagttgaa gcagtttgct 1140

actaaggcta ctgttgttgg tccagttaac taatag 1176

<210> 3

<211> 653

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atgtctgatc ctggtctcct acaggatttt tgtgtgggtg taaatgaccc tgattcagca 60

gtgtttgtaa atggaaaatt ctgcaagaac ccaaaagacg tgacaatcga cgatttctta 120

tacaaagggt ttaatattcc ctcagacaca aacaacactc aaagagcaga agccacacta 180

gtagatgtca atcgatttcc agcacttaac acattaggtg tagccatggc tcgtgtagac 240

tttgcgtcct ttggcctaaa cacacctcat ttgcaccctc gtggttctga gatattcgcg 300

gtgctagagg ggactttata tgccggcatt gtcaccaccg attacaagct tttcgacacg 360

gtgttgagaa agggtgacat gattgttttc cctcaaggct taatccactt ccagcttaat 420

cttggcaaga cagatgctct tgctattgcc tcttttggga gccaatttcc tggacgagtt 480

aatgttgcta atggtgtctt tggaactacg ccacaaattt tggatgatgt acttacccaa 540

gcgtttcagg tagatgagat ggtgattcag caacttcgat ctcagttttc aggtcaaaac 600

atatcaatca acactggaag atctattctt aaactcttaa ctgatgttgc tta 653

<210> 4

<211> 969

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atgagcgcca agtccagaac catagggatt attggagctc ctttctcaaa gggacagcca 60

cgaggagggg tggaagaagg ccctacagta ttgagaaagg ctggtctgct tgagaaactt 120

aaagaacaag agtgtgatgt gaaggattat ggggacctgc cctttgctga catccctaat 180

gacagtccct ttcaaattgt gaagaatcca aggtctgtgg gaaaagcaag cgagcagctg 240

gctggcaagg tggcacaagt caagaagaac ggaagaatca gcctggtgct gggcggagac 300

cacagtttgg caattggaag catctctggc catgccaggg tccaccctga tcttggagtc 360

atctgggtgg atgctcacac tgatatcaac actccactga caaccacaag tggaaacttg 420

catggacaac ctgtatcttt cctcctgaag gaactaaaag gaaagattcc cgatgtgcca 480

ggattctcct gggtgactcc ctgtatatct gccaaggata ttgtgtatat tggcttgaga 540

gacgtggacc ctggggaaca ctacattttg aaaactctag gcattaaata cttttcaatg 600

actgaagtgg acagactagg aattggcaag gtgatggaag aaacactcag ctatctacta 660

ggaagaaaga aaaggccaat tcatctaagt tttgatgttg acggactgga cccatctttc 720

acaccagcta ctggcacacc agtcgtggga ggtctgacat acagagaagg tctctacatc 780

acagaagaaa tctacaaaac agggctactc tcaggattag atataatgga agtgaaccca 840

tccctgggga agacaccaga agaagtaact cgaacagtga acacagcagt tgcaataacc 900

ttggcttgtt tcggacttgc tcgggagggt aatcacaagc ctattgacta ccttaaccca 960

cctaagtaa 969

<210> 5

<211> 531

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atgcagctgc accacagcaa gcaccacgcg gcctacgtga acaacctgaa cgtcaccgag 60

gagaagtacc aggaggcgtt ggccaaggga gatgttacag cccagatagc tcttcagcct 120

gcactgaagt tcaatggtgg tggtcatatc aatcatagca ttttctggac aaacctcagc 180

cctaacggtg gtggagaacc caaaggggag ttgctggaag ccatcaaacg tgactttggt 240

tcctttgaca agtttaagga gaagctgacg gctgcatctg ttggtgtcca aggctcaggt 300

tggggttggc ttggtttcaa taaggaacgg ggacacttac aaattgctgc ttgtccaaat 360

caggatccac tgcaaggaac aacaggcctt attccactgc tggggattga tgtgtgggag 420

cacgcttact accttcagta taaaaatgtc aggcctgatt atctaaaagc tatttggaat 480

gtaatcaact gggagaatgt aactgaaaga tacatggctt gcaaaaagta a 531

<210> 6

<211> 331

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

tgataggtgg tatgttttcg cttgaacttt taaatacagc cattgaacat acggttgatt 60

taataactga caaacatcac cctcttgcta aagcggccaa ggacgctgcc gccggggctg 120

tttgcgtttt tgccgtgatt tcgtgtatca ttggtttact tatttttttg ccaaagctgt 180

aatggctgaa aattcttaca tttattttac atttttagaa atgggcgtga aaaaaagcgc 240

gcgattatgt aaaatataaa gtgatagcgg taccaggagg gctggaagaa gcagaccgct 300

aacacagtac ataaaaaagg agacatgaac g 331

<210> 7

<211> 91

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

ttatctctgg cggtgttgac aagagataac aacgttgata taattgagcc cgtattgtta 60

gcatgtacgt ttaaaccagg aaacagctca t 91

<210> 8

<211> 285

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

aagaaaccaa ttgtccatat tgcatcagac attgccgtca ctgcgtcttt tactggctct 60

tctcgctaac caaaccggta accccgctta ttaaaagcat tctgtaacaa agcgggacca 120

aagccatgac aaaaacgcgt aacaaaagtg tctataatca cggcagaaaa gtccacattg 180

attatttgca cggcgtcaca ctttgctatg ccatagcatt tttatccata agattagcgg 240

atcctacctg acgcttttta tcgcaactct ctactgtttc tccat 285

<210> 9

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

tttacacttt atgcttccgg ctcgtatgtt g 31

<210> 10

<211> 1239

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

atgacgaact atcgcgttga gagtagcagc ggacgggcgg cgcgcaagac gaggctcgca 60

ttaatgggac ctgcgttcat tgcggcgatt ggttatatcg atcccggtaa ctttgcgacc 120

aatattcagg cgggtgccag cttcggctat cagctcctgt gggttgtcgt ttgggccaac 180

ctgatggcga tgctgattca gatcctctct gccaaactag ggattgccac cggtaaaaat 240

ctggcggagc aaattcgcga tcactatccg cgtcccgtag tgtggttcta ttgggttcag 300

gcagaaatta ttgcgatggc aaccgacctg gcggaattta ttggtgcggc gatcggtttt 360

aaactcattc ttggtgtttc gttgttgcaa ggtgcggtgc tgacggggat cgcgactttc 420

ctgattttaa tgctgcaacg tcgcgggcaa aaaccgctgg agaaagtgat tggcgggtta 480

ctgttgtttg ttgccgcggc ttacattgtc gagttgattt tctcccagcc taacctggcg 540

cagctgggta aaggaatggt gatcccgagt ttacctactt cggaagcggt cttcctagca 600

gcaggcgtgt taggggcgac gattatgccg catgtgattt atttgcactc ttcgctcact 660

cagcatttac atggcggttc gcgtcaacaa cgttattccg ccaccaaatg ggatgtggct 720

atcgccatga ctattgccgg ttttgtcaat ctggcgatga tggctacagc tgcggcggcg 780

ttccactttt ctggtcatac tggtgttgcc gatcttgatg aggcatatct gacgctgcaa 840

ccgttgttaa gccatgctgc ggcaacggtc tttggattaa gcctggttgc tgccggactg 900

tcctcaacgg tggtggggac actggcgggg caggtggtga tgcaggggtt cattcgcttc 960

catatcccgc tgtgggtgcg tcgtacagtc accatgttgc cgtcatttat tgtcattctg 1020

atgggattag atccgacacg gattctggtt atgagtcagg tgctgttaag ttttggtatc 1080

gccctggcgc tggttccact gctgattttc accagtgaca gcaagttgat gggcgatctg 1140

gtgaacagca aacgcgtaaa acagacaggc tgggtgattg tggtgctggt agtggcgctg 1200

aatatctggt tgttggtggg tacggcactg ggattgtaa 1239

<210> 11

<211> 90

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

tgaattcgag cactagtgca gcccgcctaa tgagcgggct tttttccatg caagctaatt 60

ccggtggaaa cgaggtcatc atttccttcc 90

<210> 12

<211> 129

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

ttatttatcg gaaggtttat cttgctgcgg tttgttgttg accatatcgc acaacataga 60

gagcagcatt aaccgtactt taaagggaga atgactaaac acgcgcatac acctcttgaa 120

ctcattcat 129

<210> 13

<211> 918

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

atgaatattc gtgatcttga gtacctggtg gcattggctg aacaccgcca ttttcggcgt 60

gcggcagatt cctgccacgt tagccagccg acgcttagcg ggcaaattcg taagctggaa 120

gatgagctgg gcgtgatgtt gctggagcgg accagccgta aagtgttgtt cacccaggcg 180

ggaatgctgc tggtggatca ggcgcgtacc gtgctgcgtg aggtgaaagt ccttaaagag 240

atggcaagcc agcagggcga gacgatgtcc ggaccgctgc acattggttt gattcccaca 300

gttggaccgt acctgctacc gcatattatc cctatgctgc accagacctt tccaaagctg 360

gaaatgtatc tgcatgaagc acagacccac cagttactgg cgcaactgga cagcggcaaa 420

ctcgattgcg tgatcctcgc gctggtgaaa gagagcgaag cattcattga agtgccgttg 480

tttgatgagc caatgttgct ggctatctat gaagatcacc cgtgggcgaa ccgcgaatgc 540

gtaccgatgg ccgatctggc aggggaaaaa ctgctgatgc tggaagatgg tcactgtttg 600

cgcgatcagg caatgggttt ctgttttgaa gccggggcgg atgaagatac acacttccgc 660

gcgaccagcc tggaaactct gcgcaacatg gtggcggcag gtagcgggat cactttactg 720

ccagcgctgg ctgtgccgcc ggagcgcaaa cgcgatgggg ttgtttatct gccgtgcatt 780

aagccggaac cacgccgcac tattggcctg gtttatcgtc ctggctcacc gctgcgcagc 840

cgctatgagc agctggcaga ggccatccgc gcaagaatgg atggccattt cgataaagtt 900

ttaaaacagg cggtttaa 918

<210> 14

<211> 641

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

atgaatatca ctgctactgt tcttcttgcg tttggtatgt cgatggatgc atttgctgca 60

tcaatcggta aaggtgccac cctccataaa ccgaaatttt gttgacaatt aatcatcggc 120

atagtatatc ggcatagtat aatacgacaa ggtgaggaac taaaccatgg ccaagttgac 180

cagtgccgtt ccggtgctca ccgcgcgcga cgtcgccgga gcggtcgagt tctggaccga 240

ccggctcggg ttctcccggg acttcgtgga ggacgacttc gccggtgtgg tccgggacga 300

cgtgaccctg ttcatcagcg cggtccagga ccaggtggtg ccggacaaca ccctggcctg 360

ggtgtgggtg cgcggcctgg acgagctgta cgccgagtgg tcggaggtcg tgtccacgaa 420

cttccgggac gcctccgggc cggccatgac cgagatcggc gagcagccgt gggggcggga 480

gttcgccctg cgcgacccgg ccggcaactg cgtgcacttc gtggccgagg agcaggactg 540

actttatcgg ctcaattatt gggaaaaaag cggaaattct cggcgggctg gtgctgatcg 600

gcatcggcgt ccagatcctc tggacgcact tccacggtta a 641

<210> 15

<211> 641

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

atgagtcgtc gcgcaggtac gccaacagca aaaaaagtga cgcagttagt gaacgtggaa 60

gagcacgttg aagggttccg ccaggtcaga gaggcgcatc gttgacaatt aatcatcggc 120

atagtatatc ggcatagtat aatacgacaa ggtgaggaac taaaccatgg ccaagttgac 180

cagtgccgtt ccggtgctca ccgcgcgcga cgtcgccgga gcggtcgagt tctggaccga 240

ccggctcggg ttctcccggg acttcgtgga ggacgacttc gccggtgtgg tccgggacga 300

cgtgaccctg ttcatcagcg cggtccagga ccaggtggtg ccggacaaca ccctggcctg 360

ggtgtgggtg cgcggcctgg acgagctgta cgccgagtgg tcggaggtcg tgtccacgaa 420

cttccgggac gcctccgggc cggccatgac cgagatcggc gagcagccgt gggggcggga 480

gttcgccctg cgcgacccgg ccggcaactg cgtgcacttc gtggccgagg agcaggactg 540

aggaaatcgc ccgtcgcgac gcggaaggca tggagcacca tgttagtgaa gagacgctcg 600

acgcttttcg tttgtttacc cagaaacacg gtgccaaatg a 641

<210> 16

<211> 452

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 16

Met Ala Pro Ala Pro Ser Ser Ala Ala Ser Ser Ile Val Val Ser Ala

1 5 10 15

Thr Ser Ser Ser Thr Val Ser Ser Ala Pro Val Ser Val Ser Ser Phe

20 25 30

Leu Pro Thr Thr Ser Ile Ala Ala Ala Thr Pro Ser Ser Ile Ala Val

35 40 45

Ala Leu Ser Ser Thr Ala Thr Val Pro Phe Ile Asp Leu Asn Pro Asn

50 55 60

Gly Pro Leu Trp Asp Pro Ser Val Ser Gly Val Pro Gln Ala Glu Arg

65 70 75 80

Gly Ser Leu Gly Ala Thr Ile Met Gly Pro Thr Asp Val Asp Thr Thr

85 90 95

Lys Ala Asn Pro Asp Leu Leu Ala Pro Pro Thr Thr Asp His Gly Ser

100 105 110

Val Asp Asn Ala Lys Trp Ala Phe Ser Leu Ser His Asn Arg Leu Gln

115 120 125

Thr Gly Gly Trp Ala Arg Glu Gln Asn Ile Gly Ala Met Pro Ile Ala

130 135 140

Thr Glu Met Ala Ser Val Asn Met Arg Leu Glu Pro Gly Ala Ile Arg

145 150 155 160

Glu Leu His Trp His Lys Thr Ala Glu Trp Ala Tyr Val Leu Lys Gly

165 170 175

Asn Thr Gln Val Thr Ala Val Asp Gln Asn Gly Lys Asn Phe Ile Gly

180 185 190

Thr Val Gly Pro Gly Asp Leu Trp Tyr Phe Pro Pro Gly Ile Pro His

195 200 205

Ser Leu Gln Ala Thr Gly Asp Asp Pro Glu Gly Ser Glu Phe Ile Leu

210 215 220

Val Phe Asp Ser Gly Ala Phe Ser Glu Asp Ser Thr Phe Leu Leu Thr

225 230 235 240

Asp Trp Met Ser His Val Pro Val Glu Val Leu Ala Lys Asn Phe Gln

245 250 255

Thr Asp Ile Ser Ala Phe Ala Arg Ile Pro Ala Glu Glu Leu Tyr Ile

260 265 270

Phe Pro Ala Ala Val Pro Pro Asp Ser Gln Gln Asp Pro Thr Ser Pro

275 280 285

Glu Gly Thr Val Pro Asn Pro Phe Thr Phe Ala Leu Ser Lys Val Pro

290 295 300

Pro Met Gln Leu Ser Gly Gly Thr Ala Lys Ile Val Asp Ser Thr Thr

305 310 315 320

Phe Thr Val Ser Lys Ala Ile Ala Ala Ala Glu Val Thr Ile Glu Pro

325 330 335

Gly Ala Ile Arg Glu Leu His Trp His Pro Thr Gln Asp Glu Trp Ser

340 345 350

Phe Phe Ile Glu Gly Arg Ala Arg Met Thr Ile Phe Ala Ala Gln Ser

355 360 365

Asn Ala Arg Thr Phe Asp Tyr Gln Ala Gly Asp Ile Gly Tyr Val Pro

370 375 380

Ala Thr Met Gly His Tyr Val Glu Asn Ile Gly Asn Thr Thr Val Arg

385 390 395 400

Tyr Leu Glu Ile Phe Asn Thr Ala Val Phe Glu Asp Ile Ser Leu Ser

405 410 415

Asn Trp Leu Ala Leu Thr Pro Pro Glu Leu Val Lys Ala His Leu Gly

420 425 430

Phe Asp Asp Ala Thr Met Ala His Leu Ala Lys Val Lys Pro Ile Val

435 440 445

Val Gly Pro Ala

450

<210> 17

<211> 389

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 17

Asn Glu Thr Ser Asp Pro Ala Leu Val Lys Pro Glu Arg Asn Gln Leu

1 5 10 15

Gly Ala Thr Ile Gln Gly Pro Asp Asn Leu Pro Ile Asp Leu Gln Asn

20 25 30

Pro Asp Leu Leu Ala Pro Pro Thr Thr Asp His Gly Phe Val Gly Asn

35 40 45

Ala Lys Trp Pro Phe Ser Phe Ser Lys Gln Arg Leu Gln Thr Gly Gly

50 55 60

Trp Ala Arg Gln Gln Asn Glu Val Val Leu Pro Leu Ala Thr Asn Leu

65 70 75 80

Ala Cys Thr Asn Met Arg Leu Glu Ala Gly Ala Ile Arg Glu Leu His

85 90 95

Trp His Lys Asn Ala Glu Trp Ala Tyr Val Leu Lys Gly Ser Thr Gln

100 105 110

Ile Ser Ala Val Asp Asn Glu Gly Arg Asn Tyr Ile Ser Thr Val Gly

115 120 125

Pro Gly Asp Leu Trp Tyr Phe Pro Pro Gly Ile Pro His Ser Leu Gln

130 135 140

Ala Thr Ala Asp Asp Pro Glu Gly Ser Glu Phe Ile Leu Val Phe Asp

145 150 155 160

Ser Gly Ala Phe Asn Asp Asp Gly Thr Phe Leu Leu Thr Asp Trp Leu

165 170 175

Ser His Val Pro Met Glu Val Ile Leu Lys Asn Phe Arg Ala Lys Asn

180 185 190

Pro Ala Ala Trp Ser His Ile Pro Ala Gln Gln Leu Tyr Ile Phe Pro

195 200 205

Ser Glu Pro Pro Ala Asp Asn Gln Pro Asp Pro Val Ser Pro Gln Gly

210 215 220

Thr Val Pro Leu Pro Tyr Ser Phe Asn Phe Ser Ser Val Glu Pro Thr

225 230 235 240

Gln Tyr Ser Gly Gly Thr Ala Lys Ile Ala Asp Ser Thr Thr Phe Asn

245 250 255

Ile Ser Val Ala Ile Ala Val Ala Glu Val Thr Val Glu Pro Gly Ala

260 265 270

Leu Arg Glu Leu His Trp His Pro Thr Glu Asp Glu Trp Thr Phe Phe

275 280 285

Ile Ser Gly Asn Ala Arg Val Thr Ile Phe Ala Ala Gln Ser Val Ala

290 295 300

Ser Thr Phe Asp Tyr Gln Gly Gly Asp Ile Ala Tyr Val Pro Ala Ser

305 310 315 320

Met Gly His Tyr Val Glu Asn Ile Gly Asn Thr Thr Leu Thr Tyr Leu

325 330 335

Glu Val Phe Asn Thr Asp Arg Phe Ala Asp Val Ser Leu Ser Gln Trp

340 345 350

Leu Ala Leu Thr Pro Pro Ser Val Val Gln Ala His Leu Asn Leu Asp

355 360 365

Asp Glu Thr Leu Ala Glu Leu Lys Gln Phe Ala Thr Lys Ala Thr Val

370 375 380

Val Gly Pro Val Asn

385

<210> 18

<211> 217

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 18

Met Ser Asp Pro Gly Leu Leu Gln Asp Phe Cys Val Gly Val Asn Asp

1 5 10 15

Pro Asp Ser Ala Val Phe Val Asn Gly Lys Phe Cys Lys Asn Pro Lys

20 25 30

Asp Val Thr Ile Asp Asp Phe Leu Tyr Lys Gly Phe Asn Ile Pro Ser

35 40 45

Asp Thr Asn Asn Thr Gln Arg Ala Glu Ala Thr Leu Val Asp Val Asn

50 55 60

Arg Phe Pro Ala Leu Asn Thr Leu Gly Val Ala Met Ala Arg Val Asp

65 70 75 80

Phe Ala Ser Phe Gly Leu Asn Thr Pro His Leu His Pro Arg Gly Ser

85 90 95

Glu Ile Phe Ala Val Leu Glu Gly Thr Leu Tyr Ala Gly Ile Val Thr

100 105 110

Thr Asp Tyr Lys Leu Phe Asp Thr Val Leu Arg Lys Gly Asp Met Ile

115 120 125

Val Phe Pro Gln Gly Leu Ile His Phe Gln Leu Asn Leu Gly Lys Thr

130 135 140

Asp Ala Leu Ala Ile Ala Ser Phe Gly Ser Gln Phe Pro Gly Arg Val

145 150 155 160

Asn Val Ala Asn Gly Val Phe Gly Thr Thr Pro Gln Ile Leu Asp Asp

165 170 175

Val Leu Thr Gln Ala Phe Gln Val Asp Glu Met Val Ile Gln Gln Leu

180 185 190

Arg Ser Gln Phe Ser Gly Gln Asn Ile Ser Ile Asn Thr Gly Arg Ser

195 200 205

Ile Leu Lys Leu Leu Thr Asp Val Ala

210 215

<210> 19

<211> 322

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 19

Met Ser Ala Lys Ser Arg Thr Ile Gly Ile Ile Gly Ala Pro Phe Ser

1 5 10 15

Lys Gly Gln Pro Arg Gly Gly Val Glu Glu Gly Pro Thr Val Leu Arg

20 25 30

Lys Ala Gly Leu Leu Glu Lys Leu Lys Glu Gln Glu Cys Asp Val Lys

35 40 45

Asp Tyr Gly Asp Leu Pro Phe Ala Asp Ile Pro Asn Asp Ser Pro Phe

50 55 60

Gln Ile Val Lys Asn Pro Arg Ser Val Gly Lys Ala Ser Glu Gln Leu

65 70 75 80

Ala Gly Lys Val Ala Glu Val Lys Lys Asn Gly Arg Ile Ser Leu Val

85 90 95

Leu Gly Gly Asp His Ser Leu Ala Ile Gly Ser Ile Ser Gly His Ala

100 105 110

Arg Val His Pro Asp Leu Gly Val Ile Trp Val Asp Ala His Thr Asp

115 120 125

Ile Asn Thr Pro Leu Thr Thr Thr Ser Gly Asn Leu His Gly Gln Pro

130 135 140

Val Ser Phe Leu Leu Lys Glu Leu Lys Gly Lys Ile Pro Asp Val Pro

145 150 155 160

Gly Phe Ser Trp Val Thr Pro Cys Ile Ser Ala Lys Asp Ile Val Tyr

165 170 175

Ile Gly Leu Arg Asp Val Asp Pro Gly Glu His Tyr Ile Leu Lys Thr

180 185 190

Leu Gly Ile Lys Tyr Phe Ser Met Thr Glu Val Asp Arg Leu Gly Ile

195 200 205

Gly Lys Val Met Glu Glu Thr Leu Ser Tyr Leu Leu Gly Arg Lys Lys

210 215 220

Arg Pro Ile His Leu Ser Phe Asp Val Asp Gly Leu Asp Pro Ser Phe

225 230 235 240

Thr Pro Ala Thr Gly Thr Pro Val Val Gly Gly Leu Thr Tyr Arg Glu

245 250 255

Gly Leu Tyr Ile Thr Glu Glu Ile Tyr Lys Thr Gly Leu Leu Ser Gly

260 265 270

Leu Asp Ile Met Glu Val Asn Pro Ser Leu Gly Lys Thr Pro Glu Glu

275 280 285

Val Thr Arg Thr Val Asn Thr Ala Val Ala Ile Thr Leu Ala Cys Phe

290 295 300

Gly Leu Ala Arg Glu Gly Asn His Lys Pro Ile Asp Tyr Leu Asn Pro

305 310 315 320

Pro Lys

<210> 20

<211> 176

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 20

Met Gln Leu His His Ser Lys His His Ala Ala Tyr Val Asn Asn Leu

1 5 10 15

Asn Val Thr Glu Glu Lys Tyr Gln Glu Ala Leu Ala Lys Gly Asp Val

20 25 30

Thr Ala Gln Ile Ala Leu Gln Pro Ala Leu Lys Phe Asn Gly Gly Gly

35 40 45

His Ile Asn His Ser Ile Phe Trp Thr Asn Leu Ser Pro Asn Gly Gly

50 55 60

Gly Glu Pro Lys Gly Glu Leu Leu Glu Ala Ile Lys Arg Asp Phe Gly

65 70 75 80

Ser Phe Asp Lys Phe Lys Glu Lys Leu Thr Ala Ala Ser Val Gly Val

85 90 95

Gln Gly Ser Gly Trp Gly Trp Leu Gly Phe Asn Lys Glu Arg Gly His

100 105 110

Leu Gln Ile Ala Ala Cys Pro Asn Gln Asp Pro Leu Gln Gly Thr Thr

115 120 125

Gly Leu Ile Pro Leu Leu Gly Ile Asp Val Trp Glu His Ala Tyr Tyr

130 135 140

Leu Gln Tyr Lys Asn Val Arg Pro Asp Tyr Leu Lys Ala Ile Trp Asn

145 150 155 160

Val Ile Asn Trp Glu Asn Val Thr Glu Arg Tyr Met Ala Cys Lys Lys

165 170 175

Claims (3)

1. The escherichia coli expression system of the manganese ion-containing recombinant protein is characterized in that a recombinant expression plasmid in the escherichia coli expression system comprises an escherichia coli molecular chaperone gene, a manganese ion-containing enzyme protein gene and an overexpression manganese ion channel-related protein gene; the manganese ion channel related protein gene is an OxyR gene, or an OxyR gene and an MntS gene;

when the escherichia coli expression system induces and expresses manganese ion-containing enzyme protein, the molecular chaperone gene and the manganese ion-containing enzyme protein gene are overexpressed, and the overexpression promotes the pumping of manganese ions into related channel protein; the original host strain of the escherichia coli expression system is Origami2(DE3) strain; the culture medium in the Escherichia coli expression system is a JL culture medium, and the JL culture medium comprises yeast extract 0.75% w/v, tryptone 1.5% w/v, KH₂PO₄ 15 mM，(NH₄)₂SO₄25mM, mannitol 1.8% w/v, sodium succinate 20mM, MgSO₄0.25mM, initial pH 6.5; and supplementing the medium with IPTG at a final concentration of 0.5mM and MnCl at a final concentration of 5mM₂Or MnSO₄；

The enzyme protein gene containing manganese ions is oxalate decarboxylase gene A2 shown as a sequence SEQ ID NO. 1.

2. The E.coli expression system of claim 1, wherein the chaperone gene is any one or more of dnaK-dnaJ-grpE, groES-groEL-tig or tig.

3. The escherichia coli expression system of claim 1, wherein the same or different medium-strength promoter or weak promoter is used as the promoter before the chaperone gene or manganese ion channel-associated protein gene; the promoter sequence is any one of a P43 promoter sequence shown as a sequence SEQ ID NO.6, or an M1-93 promoter shown as a sequence SEQ ID NO.7, or an araBAD promoter shown as a sequence SEQ ID NO.8, or a Lac promoter shown as a sequence SEQ ID NO. 9.

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