CN113736813A - Vector and method for expressing L-aspartic acid-alpha-decarboxylase by recombinant escherichia coli - Google Patents

Abstract

The invention provides a vector for expressing L-aspartate-alpha-decarboxylase in recombinant escherichia coli, and a method for expressing the L-aspartate-alpha-decarboxylase by using the corresponding vector. The method can reduce the formation of inclusion body products and ensure the expression quantity of target protein. Is suitable for industrial large-scale production and reduces the cost.

Description

Vector and method for expressing L-aspartic acid-alpha-decarboxylase by recombinant escherichia coli

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for reducing inclusion bodies when recombinant escherichia coli expresses L-aspartic acid-alpha-decarboxylase.

Background

Beta-alanine (3-aminopropionic acid) is widely applied to the fields of medicine, food, chemical industry and the like, and acrylonitrile or beta-aminopropionitrile is mainly used as a substrate for chemical synthesis at present. With the growing concern over environmental issues, traditional chemical processes are gradually being replaced by biological processes, including enzymatic conversion, whole cell catalysis, and fermentation.

At present, the synthesis of beta-alanine is mainly based on a chemical method, and the chemical synthesis method is the main method for producing beta-alanine at present, but has the defects of harsh process conditions, high energy consumption, environment friendliness and the like.

The biological enzyme method is characterized in that L-aspartic acid is used as a substrate, L-aspartic acid-alpha-decarboxylase (ADC) is added, and beta-alanine is generated through decarboxylation. L-aspartic acid-alpha-decarboxylase derived from bacteria has been studied more often, and includes those derived from Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, and the like. However, most of the bacterial L-aspartate-alpha-decarboxylase is small molecular protein, the enzyme activity is not stable enough, the recombinant Escherichia coli expressed L-aspartate-alpha-decarboxylase small molecular target protein derived from microorganisms has catalytic ability only by generating active subunits through subsequent protein self-shearing, and the small molecular protein is easily degraded by protease in the fermentation process, so that the biosynthesis method cannot be industrialized.

The insect Tribolium castaneum (Tribolium castaneum) is a commonly used source of L-aspartate-alpha-decarboxylase, but the L-aspartate-alpha-decarboxylase gene derived from Tribolium castaneum is expressed in Escherichia coli to easily form inclusion bodies with protein misfolding.

Patents CN 108660102a and CN 11110561C mention methods of reducing inclusion bodies of target proteins, co-expressing other soluble proteins, and inhibiting inclusion bodies of target proteins by other soluble proteins. Although the protein plays a certain role in inhibiting inclusion bodies, the inventor tries to construct a molecular chaperone protein with stronger solubility and a common fusion tag protein and then co-expresses the molecular chaperone protein and the common fusion tag protein with a target protein to find that: the target protein is reduced in inclusion body and the expression amount of the target protein is also reduced. The reason for the analysis is that other soluble proteins, although assisting the reduction of inclusion bodies of the target protein, occupy about half of the protein expression space. This is also a contradiction of expression currently using E.coli: it is difficult to ensure that the expression level of the protein is not reduced while the inclusion body is reduced.

In the prior art, the following methods are commonly used for solving the inclusion body:

firstly, the culture and induction temperature is reduced, the protein expression rate is reduced, the target protein is correctly folded, and an inclusion body is not easy to form. However, the growth rate of escherichia coli after cooling is greatly influenced, the biomass of the final product is reduced, more energy consumption is required for cooling in industrial large-scale production, and the cost is higher.

Secondly, constructing molecular chaperone protein with stronger solubility and common fusion tag protein, and co-expressing the molecular chaperone protein and the common fusion tag protein with the target protein. The method effectively reduces the inclusion body on the basis of not reducing the temperature, but the expression quantity of the target protein is partially occupied by the molecular chaperone protein and the common fusion tag protein, and the expression quantity of the target protein is reduced. Therefore, the method cannot ensure the expression level of the target protein while reducing the inclusion bodies.

Third, certain chemicals (e.g., antioxidants and reducing agents, etc.) are added to reduce inclusion bodies upon E.coli induction. The method has the disadvantage that many chemical substances have inhibition effects on the growth of escherichia coli and the expression of proteins.

Therefore, there is still a need in the art for a method for reducing inclusion bodies of a target protein without reducing the expression level of the target protein and without adding chemicals or increasing the energy consumption of fermentation.

Disclosure of Invention

The invention provides a method for reducing target protein inclusion bodies, not reducing the expression level of the target protein, and not needing to add chemical substances or increase the energy consumption of fermentation.

The invention selects L-aspartic acid-alpha-decarboxylase genes derived from insect tribolium castaneum, and the genes are respectively inserted into escherichia coli vectors pET32a (ampicillin resistance) and pCDFDuet-1 (streptomycin resistance) containing different resistances, so as to obtain plasmids pET32a-ADC and pCDFDuet-ADC containing target genes. Meanwhile, the two plasmids are respectively transferred into a host bacterium BL21(DE3) to obtain a co-expression double plasmid strain pET32a-pCDFDuet-ADC-BL21(DE 3). Since pCDFDuet-1 is a co-expression vector, both plasmids can coexist, and it was found that, after the expression of the protein was induced by fermentation of the obtained co-expressed dual plasmid strain: the inclusion bodies of the target protein are greatly reduced, but the total amount of the target protein is not reduced. Based on this principle, plasmids pET32a-ADC, pET28a-ADC and pCDFDuet-ADC containing the target gene were similarly obtained at the same time. Meanwhile, the three sections of plasmids are respectively transferred into a host bacterium BL21(DE3) to obtain a co-expression three-plasmid strain pET28a-pET32a-pCDFDuet-ADC-BL21(DE3), and the results are found after the obtained co-expression three-plasmid strain is fermented to induce protein expression: the inclusion body of the target protein is greatly reduced and is less than that of a double-plasmid inclusion body, but the total amount of the soluble target protein is slightly less than that of the double-plasmid co-expression.

The method successfully solves the technical problems of reducing the inclusion bodies and ensuring the expression quantity of the target protein. The reason for this may be the existence of two different resistant plasmids, which have a competitive effect in expressing the protein, and the expression rate of the target protein is reduced, so that the inclusion bodies are also reduced; and the target protein is expressed by both plasmids, so that the expression quantity of the target protein obtains a certain superposition effect. Therefore, the method successfully solves the contradiction between the total amount of the inclusion body and the target protein.

In the context of the present invention, the term "inclusion body" means that when a foreign gene is expressed in prokaryotic cells, especially in E.coli, high-density, insoluble protein particles enveloped by a membrane are formed, which are high refractive regions when observed under a microscope, and are clearly distinguished from other components in cytoplasm. Inclusion body formation is complex, related to the rate of protein production in the cytoplasm, and the newly produced polypeptide is in high concentration and does not have sufficient time to fold, thereby forming aggregates of amorphous, amorphous protein. In addition, the formation of inclusion bodies is considered to be related to the culture conditions of the host bacteria, such as medium composition, temperature, pH, ionic strength, and the like. Biologically active proteins in cells often exist in soluble or molecular complexes, and functional proteins always fold into specific three-dimensional structural forms. The proteins in inclusion bodies are aggregates in unfolded state and have no biological activity.

The terms "comprising," "including," "containing," "having," and corresponding synonyms, as used herein, mean "including, but not limited to. The term "consisting essentially of … …" means that the combination, method, etc., can include other ingredients and/or steps, so long as the other ingredients and/or steps do not materially alter the corresponding combination or method and its characteristics.

As used herein, "a" or "an", "the" and "said" include "plural" versions of the respective referents. For example, a plurality or a plurality of them may be included, and a mixed form of the plurality is also included.

The term "expression" refers to the conversion of sequence information into the corresponding expression product, including direct transcription products (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozymes, structural RNAs, or any other type of RNA) or proteins produced by mRNA translation.

The term "encoding" refers to RNA (mRNA) in which a DNA polynucleotide sequence is transcribed into a translated protein; or RNA that can be transcribed into a non-translated protein (non-coding RNA such as tRNA and rRNA); or an RNA polynucleotide sequence that can be translated into a protein.

The terms "DNA", "RNA" or "nucleic acid fragment" or "polynucleotide" refer to any one or more nucleic acid segments present in a polynucleotide or construct. The nucleic acid or fragment thereof may be provided in linear (e.g., mRNA) or circular (e.g., plasmid) form, as well as in double-stranded or single-stranded form. An "isolated" nucleic acid or polynucleotide refers to a nucleic acid molecule, DNA or RNA, which has been isolated from its natural environment. For example, in the context of the present invention, the recombinant polynucleotide contained in the vector is isolated. Other examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or purified polynucleotides in solution, and the like.

"Gene" refers to a polynucleotide comprising nucleotides that encode functional molecules, including functional molecules produced by transcription alone (biologically active RNAs) or by transcription and translation (e.g., polypeptides). The term "gene" encompasses cDNA and genomic DNA nucleic acids, and also refers to nucleic acid fragments that express a particular RNA, protein, or polypeptide, which comprise regulatory sequences preceding (5 'non-coding sequences) and following (3' non-coding sequences) the coding sequence. "native gene" refers to any gene found in nature that has its own regulatory sequences. "chimeric gene" refers to any gene that is not a native gene, comprising non-native, co-existing regulatory and/or coding sequences. "endogenous gene" refers to a native gene that is located in its natural location in the genome of an organism. "foreign gene" or "heterologous gene" refers to a gene that is not normally in a host but is introduced into the host by gene transfer.

The terms "polypeptide", "peptide", and "protein" are used interchangeably herein to refer to a polymer of amino acid residues.

As used herein, the term "recombinant" nucleic acid refers to a non-naturally occurring nucleic acid, such as by human intervention to join originally separate nucleic acid fragments together to produce a combined nucleic acid fragment having a function of interest. Artificial combination is often accomplished by chemical synthetic means or by artificial manipulation of segments of nucleic acids, for example by genetic engineering techniques.

The term "vector" refers to a self-replicating nucleic acid molecule, such as a plasmid, phage, virus, or cosmid, that is used to transfer a foreign nucleic acid fragment into a recipient cell by genetic engineering techniques. An "expression vector" refers to a vector, plasmid, or vehicle that expresses an inserted nucleic acid sequence after transformation into a host. The cloned gene (i.e., the inserted nucleic acid sequence) is typically placed under the control of regulatory elements (e.g., promoters, minimal promoters, enhancers, etc.).

Herein, the term "plasmid" refers to an extrachromosomal element, which often carries genes that are not part of the cell's core metabolic machinery, and is usually in the form of a circular double stranded DNA molecule. These elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear, circular or supercoiled single-or double-stranded DNA or RNA of any origin. Typically, a plasmid contains an origin of replication that is functional in a host cell (e.g., E.coli), and a selectable marker for detecting the host cell containing the plasmid. In some embodiments, the plasmid is a closed-loop DNA molecule. "Co-expression plasmid" means that different types of plasmids can be expressed in the same host bacterium.

The term "control sequences" refers to specific nucleic acid sequences present within or flanking (5 'or 3' to) a coding sequence that regulate transcription, RNA processing or stability, translation. Regulatory sequences include, but are not limited to: promoters, enhancers, introns, transcriptional regulatory elements, polyadenylation signals, RNA processing signals, translational enhancer elements, and the like.

The term "operably linked" refers to a regulatory element (such as, but not limited to, a promoter sequence, a transcription termination sequence, and the like) linked to a nucleic acid sequence (e.g., a coding sequence or an open reading frame) such that transcription and translation of the nucleotide sequence is controlled and regulated by the regulatory element.

The term "promoter" is a DNA regulatory region capable of binding RNA polymerase and initiating transcription of a downstream (3' direction) coding or non-coding sequence, and refers to a nucleic acid segment capable of controlling transcription of another nucleic acid segment.

"introducing" or "introducing" a cell refers to transforming a nucleic acid molecule (e.g., a plasmid, a linear nucleic acid fragment, RNA, etc.) or protein into the cell by a variety of methods including, but not limited to, Agrobacterium transformation, biolistic bombardment, electroporation, PEG transformation, etc., such that the nucleic acid or protein is capable of functioning in the cell.

First, in a first aspect of the present invention, the present invention provides a recombinant escherichia coli soluble expression system comprising at least two expression vectors, wherein each of the expression vectors comprises an L-aspartate- α -decarboxylase gene sequence, respectively, and soluble expresses L-aspartate- α -decarboxylase; wherein at least one of the expression vectors is a co-expression plasmid.

In some embodiments, wherein the L-aspartate- α -decarboxylase gene is from the insect tribolium castaneum.

In a further embodiment, the L-aspartate- α -decarboxylase gene has the nucleotide sequence as shown in SEQ ID No. 1 or a variant sequence obtained by replacing, deleting or adding one or more nucleotides to the nucleotide sequence shown in SEQ ID No. 1, wherein the variant sequence encodes a functionally identical L-aspartate- α -decarboxylase; or the L-aspartate-alpha-decarboxylase amino acid sequence is shown as SEQ ID NO. 2 or the variant sequence obtained by replacing, deleting or adding one or more amino acids in the amino acid sequence shown as SEQ ID NO. 2.

In some embodiments, the expression system wherein the expression vector is selected from the group consisting of a plasmid, a cosmid, or a phage, in preferred embodiments, the expression vector is selected from the group consisting of a plasmid.

In a further embodiment, the expression vector is a plasmid selected from two or more of the group consisting of: prokaryotic expression vectors such as pQE series (e.g., pQE2, pQE9, pQE30/31/32, pQE40, pQE70, pQE80L, pQETERs system, etc.); pET series (such as pET3a, pET3d, pET11a, pET12a, pET14b, pET15b, pET16b, pET17b, pET19b, pET20b, pET21a/b/d, pET22b, pET23b, pET24 b/b, pET25b, pET26b, pET27b, pET28 b/b, pET29 b, pET30 b, pET31b, pET32 b, pET35b, pET38b, pET39b, pET40b, pET41 b/b, pET42 b, pET43.a/b, pET44, pET49, pET b, pET302, pET303, pET 221-b, pET 4-pET 19-b, pET-b-13-19, pET-13-14-19, pET-14-III series (such as-pYb, pGpYb, pGpYcP, pG75, pYcP, pYcTpYcTpYcTpYcTpYc3672, pYcTpIII, pYcTpYcTpYcTpYcTpYcTpYcTpYcTpYcTpYcTpYcTpYcTpYcTpYcTpYcTpYcTpYcTpYcTpYcTpYcTpYcTpYcTpYcTpYcTpYcTpYcTpYcTpYcTpYcTpYcTpYcTpYcTpYcTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTpTp, pBV220/221/222, pTrx HisA/B/C, pBAD24/34/43, pBAD HisA/B/C, pPinPoint-Xa1/2/3, pGEM Ex1, pGEM7ZF (+), pTrc99A, pTwin1, pEZZ18, pkk232-8, pkk233-3, pACYC184, pBR332, pUC119, pTYB1, pTYB2, pTYB4, pTYB11, pBluescript SK (+) (), pLLP ompA, pMBP-P, pMBP-C; coli cold shock plasmid: pColdI, pColdII, pColdTF; pACYCdue-1, pETduet-1, pCDFduet-1, pRSFduet-1, pG-KJE 8. Preferably pET32a, pCDFDuet-1, pET24a, pET21a, pET28a, pACYCDuet-1, pTrcHisA.

In a preferred embodiment. Wherein the expression system is a two plasmid expression system, wherein the plasmid is selected from the group consisting of pET32a and pCDFDuet-1.

In a further preferred embodiment, wherein said L-aspartate- α -decarboxylase gene is located at the plasmid cleavage sites EcoRI/HindIII.

In another aspect of the present invention, there is also provided a host cell comprising the expression system of the present invention, wherein the host cell is a recombinant e. In a preferred embodiment, the E.coli is BL21(DE 3).

In another aspect of the invention, there is also provided the use of an expression system or an E.coli host cell as described herein for the expression of L-aspartate-alpha-decarboxylase.

In another aspect of the invention, there is also provided the use of an expression system or an E.coli host cell as described herein for reducing inclusion bodies during expression of L-aspartate-alpha-decarboxylase in E.coli.

In another aspect of the present invention, there is also provided a method for expressing an L-aspartate- α -decarboxylase protein using an e.coli expression system, comprising the steps of:

1) constructing the recombinant Escherichia coli expression system of the invention;

2) transforming an E.coli host cell with said expression system;

3) fermenting and culturing the transformed Escherichia coli host cell.

In some embodiments, the E.coli host cell is BL21(DE3), BW25113, BL21, Rosetta-origami2(DE3), BL21(DE3) -star, and the like.

In a preferred embodiment, wherein said expression method further comprises the step of 4) isolating and purifying said L-aspartate- α -decarboxylase protein.

In another aspect of the present invention, there is also provided a method for reducing inclusion bodies during the expression of L-aspartate- α -decarboxylase in Escherichia coli, comprising the steps of:

1) constructing the recombinant Escherichia coli expression system of the invention;

2) transforming an E.coli host cell with said expression system;

3) fermenting and culturing the transformed Escherichia coli host cell.

In some embodiments, the e.coli host cell is BL21(DE 3).

In a preferred embodiment, wherein said expression method further comprises the step of 4) isolating and purifying said L-aspartate- α -decarboxylase protein.

The invention has the technical effects that:

1) the inclusion body of the double-plasmid co-expression strain pET32a-pCDFDuet-BL21(DE3) is obviously less than that of pET32a-BL21(DE3) and pCDFDuet-BL21(DE 3); the amount of soluble protein of interest in the co-expressed strain was also much greater than that of pET32a-BL21(DE3) or pCDFDuet-BL21(DE 3). The enzyme activity of the co-expression strain is 95.1 percent and 123.2 percent higher than that of pET32a-BL21(DE3) and pCDFDuet-BL21(DE3) respectively. On the basis of effectively reducing the recombinant escherichia coli expression target protein inclusion body, the soluble target protein amount is improved.

2) The inclusion body of the three-plasmid co-expression strain pET28a-pET32a-pCDFDuet-BL21(DE3) is obviously less than that of the single-plasmid strain pET32a-BL21(DE3), pCDFDuet-BL21(DE3) and the two-plasmid co-expression strain pET32a-pCDFDuet-BL21(DE 3); the amount of soluble protein of interest in the three-plasmid co-expressed strain was slightly less than in the two-plasmid co-expressed strain pET32a-pCDFDuet-BL21(DE 3).

3) The method of the invention does not need cooling, and reduces energy consumption.

4) The invention does not use chemical substances to reduce the inclusion bodies, thereby not inhibiting the growth of strains and not introducing impurities for subsequent separation and purification.

Drawings

The present disclosure can be understood more readily by reference to the following figures and examples of the disclosure. Unless otherwise defined, all techniques and terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Unless otherwise indicated, the techniques employed and covered herein are standard procedures well known to those skilled in the art to which the invention pertains. The materials, methods, and examples are illustrative only and are not intended to limit the scope of the present invention in any way.

FIG. 1: a pET32a plasmid map;

FIG. 2: a pET28a plasmid map;

FIG. 3: pCDFDuet-1 plasmid map;

FIG. 4: comparing electrophoretograms of a co-expression strain pET32a-pCDFDuet-ADC-BL21(DE3) and a single-plasmid strain pET32a-ADC-BL21(DE3) after fermentation;

FIG. 5: comparison of electrophoretograms after fermentation of co-expressed strain pET32a-pCDFDuet-ADC-BL21(DE3) and single plasmid strain pCDFDuet-ADC-BL21(DE 3);

FIG. 6: comparison of the electrophoretograms after fermentation of the co-expressed species pET28a-pET32a-pCDFDuet-ADC-BL21(DE3) and the two-plasmid species pET32a-pCDFDuet-ADC-BL21(DE 3).

Detailed Description

1. Experimental Material

1.1 strains and plasmids

The Escherichia coli host strain adopted in the experimental study is BL21(DE)3 (commercially available), and the host strain is preserved in liquid nitrogen in the inventor's laboratory. Expression vectors were pET28a (kanamycin resistance), pET32a (ampicillin resistance) and pCDFDuet-1 (streptomycin resistance) (available from King-Share Biotechnology Ltd.). The target gene was derived from the L-aspartic acid-alpha-decarboxylase gene (ADC) (SEQ ID NO:1) of the insect Trimerella erythropolis, and the sequence complete gene synthesis was entrusted to Kinsley Biotechnology GmbH to construct expression vectors pET32a-ADC, pET28a-ADC and pCDFDuet-ADC (constructed by Kinsley Biotechnology GmbH). After obtaining the plasmid, the Escherichia coli BL21(DE)3 is transformed simultaneously to obtain a two-plasmid co-expression strain pET32a-pCDFDuet-ADC-BL21(DE3) and a three-plasmid co-expression strain pET28a-pET32a-pCDFDuet-ADC-BL21(DE3), and the strains are preserved in liquid nitrogen.

1.2 culture Medium

LB primary seed medium: 10g/L of peptone, 10g/L of sodium chloride and 5g/L of yeast extract powder; LB solid medium: 10g/L peptone, 10g/L sodium chloride, 5g/L yeast extract powder and 20g/L agar powder; TB flask fermentation medium: 24g/L yeast extract powder, 12g/L peptone and 4g/L glycerol.

2. Plasmid transformation

2.1 preparation of host bacterium BL21(DE3) competence

2.1.1 streaking the host bacteria on an agar culture medium plate, and culturing for 16-20 hours at 37 ℃;

2.1.2 taking 1 single colony from the plate, transferring to a 250ml triangular flask containing 50ml LB culture medium, and carrying out shaking culture at 37 ℃ and 220rpm overnight;

2.1.3 to 50ml of LB medium, 1ml of 2.1.2 culture medium was added and cultured overnight, followed by shaking at 220rpm at 37 ℃ for 3 hours;

2.1.4 putting the culture solution into a pre-cooled 50ml sterile centrifuge tube, centrifuging for 7 minutes on ice at 4 ℃ and 4,000rpm, and discarding the supernatant;

2.1.5 precipitation with 10ml ice-cold CaCl₂Resuspending the solution, centrifuging at 4 ℃ and 4,000rpm for 5 minutes, and discarding the supernatant;

2.1.6 precipitation 30ml of 0.1mol/L CaCl precooled with ice are added₂Suspending the cells, carrying out ice bath for 30 minutes, centrifuging at the temperature of 4 ℃ and the rpm of 4000 for 5 minutes, and removing the supernatant;

2.1.7 with 3ml of ice-cold 0.1mol/L CaCl₂Suspending the cells, adding 1.0ml of pre-cooled 80% glycerol solution, and subpackaging into pre-cooled 1.5ml of sterile eppendorf tubes, wherein 200 mu l of each tube is frozen at the temperature of below-60 ℃ to be used as competent cells.

2.2 preparation of the two-plasmid Co-expression Strain pET32a-pCDFDuet-ADC-BL21(DE3)

Plasmids pET32a-ADC (ampicillin resistance) and pCDFDuet-ADC (streptomycin resistance) and competent cells BL21(DE)3 were taken in liquid nitrogen and placed in an ice bath immediately in preparation for the transformation step:

2.2.1 under the aseptic condition, respectively taking 1 mu L of plasmid, slowly adding the plasmid into a centrifuge tube containing 100 mu L of competent cells, rapidly carrying out ice bath for 30 minutes, and slightly taking and placing the plasmid in the operation process without shaking, wherein the competent cells are fragile and easily influence the transformation efficiency;

2.2.2 after ice bath for 30 minutes, taking out and putting into a 42 ℃ circulating water bath kettle for heat shock for 90 seconds, taking out and taking out after ice bath for 2 minutes, adding 400 mu L of LB liquid culture medium without resistance into a centrifugal tube under aseptic condition, and performing shake bed recovery culture under the culture conditions of 37 ℃, 160RPM and 60 minutes;

2.2.3 taking 50 mul of culture solution after recovery, coating LB solid medium plate with ampicillin resistance and streptomycin resistance under the aseptic condition, and culturing in an incubator overnight at 37 ℃;

2.2.4 Single colony is taken to transfer 10mL LB and added with liquid culture medium with ampicillin resistance and streptomycin resistance, cultured at 37 ℃ and 180RPM for 6 hours, and then added with 15 percent of glycerol for conservation to obtain engineering strain pET32a-pCDFDuet-ADC-BL21(DE3), and preserved by liquid nitrogen.

2.3 preparation of three plasmid Co-expression Strain pET28a-pET32a-pCDFDuet-ADC-BL21(DE3)

Plasmids pET32a-ADC (ampicillin resistance), pET28a-ADC (kanamycin resistance) and pCDFDuet-ADC (streptomycin resistance) and competent cells BL21(DE)3, taken in liquid nitrogen, were placed rapidly in an ice bath, ready for the transformation step:

2.2.1 under the aseptic condition, respectively taking 1 mu L of plasmid, slowly adding the plasmid into a centrifuge tube containing 100 mu L of competent cells, rapidly carrying out ice bath for 30 minutes, and slightly taking and placing the plasmid in the operation process without shaking, wherein the competent cells are fragile and easily influence the transformation efficiency;

2.2.2 after ice bath for 30 minutes, taking out and putting into a 42 ℃ circulating water bath kettle for heat shock for 90 seconds, taking out and taking out after ice bath for 2 minutes, adding 400 mu L of LB liquid culture medium without resistance into a centrifugal tube under aseptic condition, and performing shake bed recovery culture under the culture conditions of 37 ℃, 160RPM and 60 minutes;

2.2.3 after the recovery, taking 50 mul of culture solution, coating LB solid culture medium plate with ampicillin resistance, kanamycin resistance and streptomycin resistance under the aseptic condition, and culturing in an incubator overnight at 37 ℃;

2.2.4 Single colonies were picked, inoculated into 10mL LB liquid medium to add ampicillin, kanamycin and streptomycin resistance, cultured at 37 ℃ at 180RPM for 6 hours, and then preserved with 15% glycerol to obtain the engineered strain pET28a-ADC-pET32a-pCDFDuet-ADC-BL21(DE3), which was deposited under liquid nitrogen.

2.4 preparation of Strain PET32a-ADC-BL21(DE3)

Plasmid pET32a-ADC (ampicillin resistance) and competent cells BL21(DE)3 were taken from liquid nitrogen and placed rapidly in an ice bath, ready for the transformation step:

2.3.1 under the aseptic condition, respectively taking 1 mu L of plasmid, slowly adding the plasmid into a centrifuge tube containing 100 mu L of competent cells, rapidly carrying out ice bath for 30 minutes, and slightly taking and placing the plasmid in the operation process without shaking, wherein the competent cells are fragile and easily influence the transformation efficiency;

2.3.2 after ice bath for 30 minutes, taking out and putting into a 42 ℃ circulating water bath kettle for heat shock for 90 seconds, taking out and taking out after ice bath for 2 minutes, adding 400 mu l of LB liquid culture medium without resistance into a centrifugal tube under aseptic condition, and recovering and culturing by a shaking table under 37 ℃, 160RPM and 60 minutes;

2.3.3 after the recovery, taking 50 mul of culture solution under the aseptic condition, coating an LB solid culture medium plate added with ampicillin resistance and streptomycin resistance, and culturing in an incubator overnight at 37 ℃;

2.3.4 Single colony is taken to be transferred to 10mL LB and added with ampicillin resistant liquid culture medium, cultured for 6 hours at 37 ℃ and 180RPM, and then added with 15 percent of glycerol for preserving the strain to obtain the engineering strain pET32a-ADC-BL21(DE3), and preserved by liquid nitrogen.

2.5 preparation of Strain pCDFDuet-ADC-BL21(DE3)

Plasmid pCDFDuet-ADC (streptomycin resistant) and competent cells BL21(DE3) were taken in liquid nitrogen and placed rapidly in an ice bath, ready for the transformation step:

2.4.1 under the aseptic condition, respectively taking 1 mu L of plasmid, slowly adding the plasmid into a centrifuge tube containing 100 mu L of competent cells, rapidly carrying out ice bath for 30 minutes, and slightly taking and placing the plasmid in the operation process without shaking, wherein the competent cells are fragile and easily influence the transformation efficiency;

2.4.2 after ice bath for 30 minutes, taking out and putting into a 42 ℃ circulating water bath kettle for heat shock for 90 seconds, taking out and taking out after ice bath for 2 minutes, adding 400 mu L of LB liquid culture medium without resistance into a centrifugal tube under aseptic condition, and performing shake bed recovery culture under the culture conditions of 37 ℃, 160RPM and 60 minutes;

2.4.3 taking 50 mul of culture solution after recovery, coating LB solid medium plate with ampicillin resistance and streptomycin resistance under the aseptic condition, and culturing in an incubator overnight at 37 ℃;

2.4.4 Single colony is selected, transferred to 10mL LB, added to streptomycin resistant liquid medium, cultured at 37 deg.C and 180RPM for 6 hours, added with 15% glycerol for conservation to obtain the engineering strain pCDFDuet-ADC-BL21(DE3), and preserved in liquid nitrogen.

3. Strain shake flask fermentation

Experimental equipment: the device comprises an ultra-clean workbench, a shaking table, an incubator, a sterilization pot, a liquid transfer gun, a centrifugal machine, an ultrasonication instrument, a microwave oven, an oscillator, a protein electrophoresis apparatus, an electronic balance and a liquid nitrogen tank.

Example 1:

1) shake flask cell fermentation contrast culture of double-plasmid co-expression strain pET32a-pCDFDuet-ADC-BL21(DE3) and single-plasmid strain pET32a-ADC-BL21(DE3)

Co-expression of pET32a-pCDFDuet-ADC-BL21(DE 3): glycerol tube 100 uL → 10mL test tube LB liquid culture medium (adding 100mg/L ampicillin and 50mg/L streptomycin), 30 ℃, 150RPM overnight culture, transferring 5mL → 500mL TB shake flask fermentation culture medium liquid loading volume 200mL (adding 100mg/L ampicillin and 50mg/L streptomycin), 37 ℃, 180RPM culture for 6 hours → adding 10g/L alpha-lactose for induction, inducing temperature 30 ℃ → inducing for 17 hours, finishing centrifugation and collecting cells → taking 1g wet centrifugal cells adding 10g deionized water for mixing → ultrasonic disruption centrifugation, taking disruption supernatant and disruption liquid sediment, adding deionized water for diluting the replacement liquid ten times, and running protein electrophoresis SDS-PAGE to detect protein expression quantity respectively.

pET32a-ADC-BL21(DE 3): glycerol tube 100 uL → 10mL test tube LB liquid culture medium (adding 100mg/L ampicillin), 30 ℃, 150RPM overnight culture, transferring 5mL → 500mL LTB shake flask fermentation culture medium liquid loading amount 200mL (adding 100mg/L ampicillin), 37 ℃, 180RPM culture 6 hours → adding 10g/L alpha-lactose for induction, inducing temperature 30 ℃ → inducing 17 hours to finish centrifugal collection cells → 1g centrifugal wet cells adding 10g deionized water for mixing → ultrasonic disruption centrifugation, taking disruption supernatant and disruption liquid for precipitation, adding deionized water for diluting ten times of the replacement liquid, and running protein electrophoresis SDS-PAGE respectively to detect protein expression.

2) Determination of enzymatic Properties

Taking 100 mu L of enzyme solution, putting the enzyme solution into a 1.5mL centrifuge tube, adding L-sodium aspartate with the final concentration of 100mmol/L and the final concentration of PLP of 1mmol/L, and reacting for 30min at 37 ℃ and under the condition of pH 6.5 to detect the enzyme activity.

Definition of enzyme activity: the enzyme amount required for converting sodium L-aspartate to generate 1mmol of beta-alanine per hour is one enzyme activity unit U under the conditions of 37 ℃ and pH 6.5.

Specific enzyme activity definition: the unit number of enzyme activity contained in each gram of protein.

Example 2:

1) shake flask cell fermentation contrast culture of double plasmid co-expression strain pET32a-pCDFDuet-ADC-BL21(DE3) and single plasmid strain pCDFDuet-ADC-BL21(DE3)

pET32a-pCDFDuet-ADC-BL21(DE 3): glycerol tube 100 uL → 10mL test tube LB liquid culture medium (100 mg/L of ampicillin and 50mg/L of streptomycin are added), 30 ℃, 150RPM overnight culture, 5mL → 500mL TB flask fermentation culture medium is transferred to be filled with 200mL (100 mg/L of ampicillin and 50mg/L of streptomycin are added), 37 ℃, 180RPM culture is carried out for 6 hours → 10g/L of alpha-lactose is added for induction, the induction temperature is 30 ℃ and → induction is carried out for 17 hours, centrifugal collection cells are finished, 1g of centrifugal wet cells are added with 10g of deionized water for mixing, ultrasonic disruption centrifugation is carried out, disrupted supernatant and disrupted solution precipitate are taken, deionized water is added for dilution, and protein electrophoresis SDS-PAGE is respectively carried out to detect protein expression.

pCDFDuet-ADC-BL21(DE 3): glycerol tube 100 μ L → 10mL test tube LB liquid medium (50mg/L streptomycin), 30 ℃, 150RPM overnight culture, transfer 5mL → 500mL LTB shake flask fermentation medium liquid loading 200mL (50mg/L streptomycin), 37 ℃, 180RPM culture 6 hours → add 10g/L alpha-lactose to induce, induction temperature 30 ℃ → induction 17 hours end centrifugal collection cells → 1g centrifugal wet cells add 10g deionized water to mix, ultrasonic disruption centrifugation takes disruption supernatant and disruption liquid sediment to dilute tenfold the replacement liquid, protein electrophoresis SDS-PAGE detects protein expression.

2) Determination of enzymatic Properties

100 mu L of enzyme solution is put into a 1.5mL centrifuge tube, added with 100 mmol/LL-sodium aspartate with the final concentration of 1mmol/L PLP, and reacted for 30min at 37 ℃ and pH 6.5 to detect the enzyme activity.

Definition of enzyme activity: the enzyme amount required for converting the L-sodium aspartate to generate 1mmol of beta-alanine per hour is one enzyme activity unit U under the conditions of 37 ℃ and pH 6.5.

Example 3:

1) three plasmid co-expression strain pET28a-pET32a-pCDFDuet-ADC-BL21(DE3) and two plasmid strain pET32a-pCDFDuet-ADC-BL21(DE3) shake flask cell fermentation contrast culture

Two-plasmid co-expression of pET32a-pCDFDuet-ADC-BL21(DE 3): glycerol tube 100 uL → 10mL test tube LB liquid culture medium (adding 100mg/L ampicillin and 50mg/L streptomycin), 30 ℃, 150RPM overnight culture, transferring 5mL → 500mL TB shake flask fermentation culture medium liquid loading volume 200mL (adding 100mg/L ampicillin and 50mg/L streptomycin), 37 ℃, 180RPM culture for 6 hours → adding 10g/L alpha-lactose for induction, inducing temperature 30 ℃ → inducing for 17 hours, finishing centrifugation and collecting cells → taking 1g wet centrifugal cells adding 10g deionized water for mixing → ultrasonic disruption centrifugation, taking disruption supernatant and disruption liquid sediment, adding deionized water for diluting the replacement liquid ten times, and running protein electrophoresis SDS-PAGE to detect protein expression quantity respectively.

Three plasmids co-express pET28a-pET32a-pCDFDuet-ADC-BL21(DE 3): glycerol tube 100 μ L → 10mL test tube LB liquid culture medium (adding 100mg/L ampicillin, 50mg/L streptomycin and kanamycin), 30 ℃, 150RPM overnight culture, transferring 5mL → 500mL LTB shake flask fermentation culture medium liquid loading amount of 200mL (adding 100mg/L ampicillin, 50mg/L streptomycin and kanamycin), 37 ℃, 180RPM culture for 6 hours → adding 10g/L alpha-lactose for induction, inducing temperature of 30 ℃ → inducing for 17 hours to finish centrifugation and collecting cells → taking 1g centrifugation wet cells adding 10g deionized water for mixing → ultrasonication and centrifugation, taking the disruption supernatant and disruption solution for precipitation, adding deionized water for diluting ten times of the replacement solution, and running protein electrophoresis SDS-PAGE respectively to detect protein expression.

2) Determination of enzymatic Properties

Taking 100 mu L of enzyme solution, putting the enzyme solution into a 1.5mL centrifuge tube, adding L-sodium aspartate with the final concentration of 100mmol/L and the final concentration of PLP of 1mmol/L, and reacting for 30min at 37 ℃ and under the condition of pH 6.5 to detect the enzyme activity.

Definition of enzyme activity: the enzyme amount required for converting the L-sodium aspartate to generate 1mmol of beta-alanine per hour is one enzyme activity unit U under the conditions of 37 ℃ and pH 6.5.

4. Enzyme activity detection and SDS-PAGE electrophoresis result

The samples of examples 1 and 2 were subjected to protein electrophoresis SDS-PAGE, and the results of the number of the samples and the detection of the enzyme activity are shown below:

electrophoresis results: SDS-PAGE results of the supernatant and pellet of the disruption obtained by co-expressing pET32a-pCDFDuet-ADC-BL21(DE3) and expressing pET32a-ADC-BL21(DE3) or pCDFDuet-ADC-BL21(DE3) are shown in FIG. 4 and FIG. 5.

As can be seen from the electrophoretogram bands, the protein content in the supernatant fraction of the disruption solution co-expressing pET32a-pCDFDuet-ADC-BL21(DE3) is significantly higher than that of the disruption solution co-expressing pET32a-ADC-BL21(DE3) or pCDFDuet-ADC-BL21(DE 3); the inclusion body of target protein of coexpression strain pET32a-pCDFDuet-ADC-BL21(DE3) is obviously less than that of single plasmid strains pET32a-ADC-BL21(DE3) and pCDFDuet-ADC-BL21(DE 3).

Compared with the two-plasmid co-expression, the total amount of soluble target protein of the two-plasmid co-expression strain pET32a-pCDFDuet-ADC-BL21(DE3) is higher than that of the three-plasmid co-expression strain pET28a-pET32a-pCDFDuet-ADC-BL21(DE3), namely the co-expression effect of the two-plasmid is better than that of the three-plasmid co-expression, but the inclusion body co-expression of the three-plasmid is less than that of the two-plasmid co-expression, namely the more plasmids are, the more the effect of degrading the inclusion body is obvious, but the total protein amount is reduced.

The results show that the co-expression method of the invention really plays a role in inhibiting the inclusion body of the target protein, and more importantly, the expression quantity of the soluble target protein of the co-expression strain is more than that of the single plasmid strain, namely, the co-expression strain of the invention reduces the inclusion body and simultaneously improves the total expression quantity of the soluble target protein.

The invention skillfully inserts the same target gene into two different types of escherichia coli vectors pET32a and pCDFDuet; both types of vectors can coexist in E.coli, and the two vectors ligated with the target gene are transferred simultaneously. The target protein inclusion body is reduced, the expression quantity of the target protein is not reduced, and chemical substances are not required to be added or the fermentation energy consumption is not required to be increased.

It should be noted that, although some features of the present invention have been illustrated by the above embodiments, the present invention is not limited thereto. It will be appreciated by those skilled in the art that changes may 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.

Sequence listing

Bio-technology Co., Ltd. < 110 > Guang-an Mo Jia

Carrier and method for expressing L-aspartic acid-alpha-decarboxylase by < 120 > recombinant escherichia coli

＜130＞CID210064

＜160＞2

＜170＞PatentIn version 3.5

＜210＞1

＜211＞1623

＜212＞DNA

＜213＞Tribolium castaneum

＜400＞1

atgccggcca caggcgaaga ccaagacctg gtgcaagacc tcatcgagga gcccgccacc 60

ttcagcgacg ccgtcctctc ctccgacgag gaactcttcc accagaagtg ccccaaaccc 120

gcccccattt actccccggt ctcgaaaccg gtctccttcg agagcctccc caacaggcgc 180

ctccacgagg agttcctccg cagctcggtg gacgtcctcc tccaggaggc ggtgttcgag 240

ggaacgaacc gcaagaaccg ggtgctgcaa tggcgggagc cggaggagtt gaggcgtctg 300

atggactttg gggtgcggag tgcgccctcc acgcacgagg agttgttgga ggtgttgaag 360

aaggttgtaa cttattcggt taaaaccgga catccgtact tcgtgaacca gttgttctcg 420

gcggtggatc cgtacggttt ggtggcacaa tgggccacgg atgcgctcaa tccgagtgtt 480

tacacctacg aggtttcgcc ggtttttgtt ctgatggagg aagtggtttt gagggagatg 540

agggccattg tggggttcga ggggggaaag ggcgatggga ttttttgccc aggagggtcc 600

attgccaatg gatatgccat cagttgtgcc agatacaggt ttgtgcccga tattaagaaa 660

aaaggcctcc actctctccc ccgtttggtc ctcttcacct ctgaagatgc ccactattcc 720

atcaaaaaac tcgcctcttt ccaaggcatc ggcaccgaca acgtctactt gatacgaacg 780

gacgcccgag gtcgcatgga cgtctcgcac ctggtggagg aaatcgagcg ttcgctccgt 840

gaaggcgccg ctcctttcat ggtcagtgcc accgctggaa ccacagtgat tggtgccttt 900

gaccccatcg aaaaaatcgc agatgtgtgc caaaaataca aactgtggtt gcacgtggat 960

gccgcctggg gaggtggcgc gcttgtctct gccaaacacc gccacctcct caaagggatt 1020

gagagggccg actcggtcac ctggaaccct cacaaactcc taacagcccc ccagcaatgt 1080

tccacacttt tactgcgaca tgagggtgtc ctcgccgagg cgcattccac gaacgccgct 1140

tacctcttcc aaaaagacaa attctacgac accaaatacg acacgggcga caagcacatc 1200

cagtgcggcc gcagggccga cgtcctcaag ttctggttca tgtggaaggc gaagggaaca 1260

tcagggttgg agaaacacgt cgataaagtg ttcgaaaatg cgagattttt caccgattgt 1320

ataaaaaatc gggaagggtt tgaaatggtg atagcggagc ccgaatacac aaacatctgc 1380

ttttggtacg tgccgaagag tctgaggggg cgcaaggacg aagccgatta caaagacaag 1440

ctgcataagg tggcccccag gattaaggag aggatgatga aggagggctc catgatggtc 1500

acgtaccagg cgcaaaaggg acacccgaat tttttcagga ttgtgttcca gaattcgggg 1560

cttgacaagg ctgatatggt gcaccttgtt gaggagattg agcggttggg gagcgatctt 1620

tga 1623

＜210＞2

＜211＞540

＜212＞PRT

＜213＞Tribolium castaneum

＜400＞2

Met Pro Ala Thr Gly Glu Asp Gln Asp Leu Val Gln Asp Leu Ile Glu

1 5 10 15

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

20 25 30

Phe His Gln Lys Cys Pro Lys Pro Ala Pro Ile Tyr Ser Pro Val Ser

35 40 45

Lys Pro Val Ser Phe Glu Ser Leu Pro Asn Arg Arg Leu His Glu Glu

50 55 60

Phe Leu Arg Ser Ser Val Asp Val Leu Leu Gln Glu Ala Val Phe Glu

65 70 75 80

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

85 90 95

Leu Arg Arg Leu Met Asp Phe Gly Val Arg Ser Ala Pro Ser Thr His

100 105 110

Glu Glu Leu Leu Glu Val Leu Lys Lys Val Val Thr Tyr Ser Val Lys

115 120 125

Thr Gly His Pro Tyr Phe Val Asn Gln Leu Phe Ser Ala Val Asp Pro

130 135 140

Tyr Gly Leu Val Ala Gln Trp Ala Thr Asp Ala Leu Asn Pro Ser Val

145 150 155 160

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

165 170 175

Leu Arg Glu Met Arg Ala Ile Val Gly Phe Glu Gly Gly Lys Gly Asp

180 185 190

Gly Ile Phe Cys Pro Gly Gly Ser Ile Ala Asn Gly Tyr Ala Ile Ser

195 200 205

Cys Ala Arg Tyr Arg Phe Val Pro Asp Ile Lys Lys Lys Gly Leu His

210 215 220

Ser Leu Pro Arg Leu Val Leu Phe Thr Ser Glu Asp Ala His Tyr Ser

225 230 235 240

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

245 250 255

Leu Ile Arg Thr Asp Ala Arg Gly Arg Met Asp Val Ser His Leu Val

260 265 270

Glu Glu Ile Glu Arg Ser Leu Arg Glu Gly Ala Ala Pro Phe Met Val

275 280 285

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

290 295 300

Lys Ile Ala Asp Val Cys Gln Lys Tyr Lys Leu Trp Leu His Val Asp

305 310 315 320

Ala Ala Trp Gly Gly Gly Ala Leu Val Ser Ala Lys His Arg His Leu

325 330 335

Leu Lys Gly Ile Glu Arg Ala Asp Ser Val Thr Trp Asn Pro His Lys

340 345 350

Leu Leu Thr Ala Pro Gln Gln Cys Ser Thr Leu Leu Leu Arg His Glu

355 360 365

Gly Val Leu Ala Glu Ala His Ser Thr Asn Ala Ala Tyr Leu Phe Gln

370 375 380

Lys Asp Lys Phe Tyr Asp Thr Lys Tyr Asp Thr Gly Asp Lys His Ile

385 390 395 400

Gln Cys Gly Arg Arg Ala Asp Val Leu Lys Phe Trp Phe Met Trp Lys

405 410 415

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

420 425 430

Asn Ala Arg Phe Phe Thr Asp Cys Ile Lys Asn Arg Glu Gly Phe Glu

435 440 445

Met Val Ile Ala Glu Pro Glu Tyr Thr Asn Ile Cys Phe Trp Tyr Val

450 455 460

Pro Lys Ser Leu Arg Gly Arg Lys Asp Glu Ala Asp Tyr Lys Asp Lys

465 470 475 480

Leu His Lys Val Ala Pro Arg Ile Lys Glu Arg Met Met Lys Glu Gly

485 490 495

Ser Met Met Val Thr Tyr Gln Ala Gln Lys Gly His Pro Asn Phe Phe

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Arg Ile Val Phe Gln Asn Ser Gly Leu Asp Lys Ala Asp Met Val His

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Claims (17)

1. A recombinant escherichia coli soluble expression system, comprising at least two expression vectors, wherein each of the expression vectors comprises an L-aspartate- α -decarboxylase gene sequence, and wherein L-aspartate- α -decarboxylase is soluble; wherein at least one of the expression vectors is a co-expression plasmid.

2. The expression system of claim 1, wherein the L-aspartate-a-decarboxylase gene is from the insect tribolium castaneum.

3. The expression system of claim 2, wherein: the L-aspartate-alpha-decarboxylase gene has a nucleotide sequence shown as SEQ ID NO. 1 or a variant sequence obtained by replacing, deleting or adding one or more nucleotides in the nucleotide sequence shown as SEQ ID NO. 1, wherein the variant sequence encodes L-aspartate-alpha-decarboxylase with the same function; or

The L-aspartate-alpha-decarboxylase gene has an amino acid sequence shown as SEQ ID NO. 2 or a variant sequence obtained by replacing, deleting or adding one or more amino acids from the amino acid sequence shown as SEQ ID NO. 2.

4. The expression system according to any one of claims 1 to 3, wherein the expression vector is selected from the group consisting of a plasmid, a cosmid or a phage, preferably a plasmid.

5. The expression system of claim 4, wherein the expression vector is a plasmid selected from two or more of the group consisting of: pET32a, pCDFDuet-1, pET24a, pET21a, pET28a, pACYCDuet-1, pTrcHisA.

6. The expression system of claim 5, wherein the expression system is a two plasmid co-expression system or a three plasmid co-expression system, wherein the plasmid is selected from pET28a, pET32a, or pCDFDuet-1.

7. The expression system according to any one of claims 1 to 6, wherein the cleavage site of the L-aspartate- α -decarboxylase gene in the plasmid is EcoRI/HindIII.

8. A host cell comprising the expression system of any one of claims 1-7, wherein the host cell is a recombinant e.

9. The host cell according to claim 8, wherein the E.coli is selected from BL21(DE3), BW25113, BL21, Rosetta-origami2(DE3) or BL21(DE3) -star.

10. Use of the expression system of any one of claims 1-7, the host cell of claim 8 or 9 for expressing L-aspartate- α -decarboxylase.

11. Use of the expression system of any one of claims 1-7, the host cell of claim 8 or 9 for reducing inclusion bodies during expression of L-aspartate- α -decarboxylase in e.

12. A method for expressing L-aspartic acid-alpha-decarboxylase protein by an escherichia coli expression system is characterized by comprising the following steps:

1) constructing a recombinant E.coli expression system according to any one of claims 1 to 7;

2) transforming an E.coli host cell with said expression system;

3) fermenting and culturing the transformed Escherichia coli host cell.

13. The method of claim 12, wherein the e.coli host cell is BL21(DE 3).

14. The method according to claim 12 or 13, further comprising the step of:

4) separating and purifying the L-aspartic acid-alpha-decarboxylase protein.

15. A method for reducing inclusion bodies in the process of expressing L-aspartic acid-alpha-decarboxylase by Escherichia coli, which is characterized by comprising the following steps:

1) constructing a recombinant E.coli expression system according to any one of claims 1 to 7;

2) transforming an E.coli host cell with said expression system;

3) fermenting and culturing the transformed Escherichia coli host cell.

16. The method of claim 15, wherein the e.coli host cell is BL21(DE 3).

17. The method according to claim 15 or 16, further comprising the step of:

4) separating and purifying the L-aspartic acid-alpha-decarboxylase protein.

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