CN110607319A - An expression vector suitable for secreting and expressing proteins of Bacillus subtilis and its application - Google Patents

Abstract

The invention discloses an expression carrier suitable for secreting and expressing proteins of bacillus subtilis and its application, belonging to the fields of genetic engineering and biotechnology. The secretion vector is to connect the gene (nucleotide sequence such as SEQ ID NO.1) encoding signal peptide SP _phoD and the gene (nucleotide sequence such as SEQ ID NO.3) encoding molecular chaperone to pMA5‑P43 respectively Construction of the secretion plasmid pMA5‑SPP. Using pMA5‑SPP as a vector, L‑asparaginase was expressed in Bacillus subtilis, its extracellular secretion accounted for 38% of the total expression, and the extracellular secretion was 2.95 times that of the common signal peptide SP _pel -mediated secretion.

Description

An expression vector suitable for secreting and expressing proteins of Bacillus subtilis and its application

technical field

The invention relates to an expression carrier suitable for secreting and expressing proteins of bacillus subtilis and its application, belonging to the fields of genetic engineering and biotechnology.

Background technique

In the process of using microorganisms to express recombinant proteins, good secretion performance reduces product recovery costs and is an important consideration for whether recombinant proteins can be industrially produced. Signal peptide is a peptide chain at the N-terminal of secretory protein that can mediate protein secretion to the extracellular space, and is of great significance to the secretory expression of protein. Molecular chaperones can recognize and bind to incompletely folded or assembled proteins in cells, helping these proteins to fold and secrete (Molecular microbiology, 2010, 8(4): 727-37; Microbial cell factories, 2015, 14, 92) . Therefore, it is of great significance to select appropriate signal peptides and molecular chaperones in constructing secretory expression vectors for proteins.

Bacillus subtilis has good secretion ability due to the lack of outer cell membrane, and it is an ideal host for secreting biological products. At the same time, Bacillus subtilis also has the characteristics of food safety, clear genetic information, good production technology and fermentation basis, so it is widely used in the biological fermentation preparation of protein, food additives and antibiotics (Trends inbiotechnology, 1992, 10 (7) :247-56; Journal of clinical microbiology, 1998, 36(1): 325-6; Nature, 1997, 390(6657): 249-56.).

In the process of secreting and expressing foreign proteins with Bacillus subtilis as host bacteria, it is of great significance to construct vectors containing effective signal peptides and molecular chaperones for protein secretion. At present, there is no literature report on simultaneously constructing the signal peptide SP _phoD gene and the molecular chaperone PrsA gene on the carrier to increase the secretion level of the Bacillus subtilis protein.

There are relatively few reports on the extracellular secretion of L-asparaginase. Studies have shown that B. subtilis B11-06 L-asparaginase was expressed with Bacillus subtilis 168 as the host bacteria, and the extracellular enzyme activity accounted for the total The activity is 57.1% (JournalOf Agricultural And Food Chemistry, 2013, 61(39):9428-9434), but the activity of the recombinase is only 9.98U/mL. Using Bacillus subtilis 168 as the host bacteria, expressing L-asparaginase derived from Pyrococcus yayanosii, the extracellular and intracellular enzyme activities were 23.31U/mL and 65.72U/mL respectively (Scientific Reports, 2018, 8(1): 7915), the enzyme activity level is limited.

Therefore, providing a method for further increasing the extracellular secretion level of L-asparaginase has important application value for the industrial preparation of L-asparaginase.

Contents of the invention

The first object of the present invention is to provide a kind of bacillus subtilis expression vector, be to connect the gene of coding signal peptide SPphoD and the gene of coding molecular chaperone PrsA to carrier pMA5-P43, obtain expression vector pMA5-SPP; Said signal The amino acid sequence of the peptide SPphoD is shown in SEQ ID NO.1, and the amino acid sequence of the molecular chaperone PrsA is shown in SEQ ID NO.3. In one embodiment of the present invention, the gene encoding the signal peptide SPphoD is connected between the multiple cloning site EcoR V and Kpn I of the vector pMA5-P43, expressed under the mediation of the P43 promoter, and The gene encoding the molecular chaperone PrsA is connected between the multiple cloning sites BamH I and Mlu I of the vector pMA5-P43, and expressed under the mediation of the PpHpaII promoter.

The pMA5-P43 plasmid is obtained by linking the promoter P43 to the pMA5 vector by enzyme-cut ligation.

The construction method of the pMA5-P43 plasmid specifically uses forward primer F (nucleotide sequence shown in SEQ ID NO.15) and reverse primer R (nucleotide sequence shown in SEQ ID NO.16) from The promoter P43 was amplified in the genome of Bacillus subtilis (Bacillus subtilis) 168; the vector pMA5 was double-digested with restriction endonucleases EcoR I and Hind III; the homologous recombination kit ( MultiS One-Step Cloning Kit, Novozyme) The promoter P43 was connected between the multiple cloning sites EcoR I and Hind III of pMA5 to obtain the recombinant plasmid pMA5-P43.

In one embodiment of the present invention, the nucleotide sequence of the gene encoding the signal peptide SPphoD is shown in SEQ ID NO.2.

In one embodiment of the present invention, the nucleotide sequence of the gene encoding molecular chaperone PrsA is shown in SEQ ID NO.4.

The second object of the present invention is to provide a genetically engineered bacterium using the above expression vector as the expression vector.

In one embodiment of the present invention, the genetically engineered bacteria use Bacillus subtilis as a host.

In one embodiment of the present invention, the genetically engineered bacterium expresses L-asparaginase whose amino acid sequence is shown in SEQ ID NO.13.

In one embodiment of the present invention, the nucleotide sequence of the gene encoding the L-asparaginase is shown in SEQ ID NO.14.

The third object of the present invention is to provide the preparation method of the above-mentioned genetically engineered bacteria, which is to connect the gene encoding L-asparaginase between the restriction enzyme sites Kpn I and Hind III of pMA5-SPP to obtain recombinant Plasmid, the recombinant plasmid is transformed into Bacillus subtilis 168 to obtain the Bacillus subtilis genetically engineered bacterium.

The fourth object of the present invention is to provide a method for preparing L-asparaginase, which is to inoculate the above-mentioned genetically engineered bacteria in LB medium, 35-39 ° C, 200-220rpm, cultivate 8-12h, press 0.5 -5% of the inoculum was transferred to new LB medium, cultured at 35-39°C for 20-30 hours, and the fermentation broth was centrifuged. The supernatant was the extracellular crude enzyme solution, and the cell broken supernatant was the intracellular crude enzyme solution. .

The fifth object of the present invention is to provide the application of the above Bacillus subtilis expression vector in the preparation of endogenous or exogenous protein.

The present invention uses the signal peptide SPphoD to mediate protein secretion, and co-expresses the molecular chaperone PrsA to assist the protein to transport out of the cell membrane, and uses the L-asparaginase extracellular secretion to reach 38% of the total expression, which is mediated by the common signal peptide SPpel Compared with secretory expression, the method provided by the invention increases the extracellular secretion of L-asparaginase by 2.95 times.

Detailed ways

Example 1 Construction of expression vector pMA5-SPP

(1) Amplification of SP _phoD and PrsA genes: with the genome of Bacillus subtilis 168 as a template, F1primer (nucleotide sequence shown in SEQ ID NO.5) and R1 primer (nucleotide sequence shown in SEQ ID NO.5) and R1 primer (nucleotide sequence shown in SEQ ID NO. NO.6), F2 primer (nucleotide sequence as shown in SEQ ID NO.7) and R2 primer (nucleotide sequence as shown in SEQ ID NO.8) were primers to clone SP _phoD and PrsA genes respectively .

(2) On the basis of the vector pMA5-P ₄₃ , the homologous recombination kit ( MultiS One-Step Cloning Kit (Novazyme) connects the signal peptide SP _phoD gene between its multiple cloning sites EcoR V and Kpn I, and connects the molecular chaperone PrsA gene to its multiple cloning sites BamH I and Mlu I In between, the expression vector pMA5-SPP, which is beneficial to protein secretion, was constructed.

Example 2 Construction of L-asparaginase secretion expression strain

Using the plasmid pMA5-pyasnase (see the literature for the construction method: Xu L, Xian Z, Shuqin X, et al. Simultaneous cell disruption and semi-quantitative activity assays for high-throughput screening of thermostable L-asparaginases [J]. Scientific Reports, 2018, 8( 1): 7915.) as a template, with F3 primer (shown in the nucleotide sequence of SEQ ID NO.9) and R4 primer (shown in the nucleotide sequence of SEQ ID NO.10) as primers to amplify the coding L-day The gene (nucleotide sequence shown in SEQ ID NO.14) of paraginase, and homologous recombination kit ( MultiS One-Step Cloning Kit) to connect the gene between the restriction enzyme sites Kpn I and Hind III of pMA5-SPP to obtain a recombinant plasmid. The obtained recombinant plasmid was chemically transformed into Bacillus subtilis 168 competent cells, and the L-asparaginase secretion expression strain B.subtilis/pMA5-SPP-pyasnase was constructed

Example 3 Determination of B. subtilis/pMA5-SPP-pyasnase secretion

(1) Combine the recombinant bacteria B.subtilis/pMA5-SPP-pyasnase obtained in Example 2 with the L-asparaginase expression strain mediated only by common SPpel (the construction method is the same as in Examples 1 and 2, and the primer sequences are as SEQ shown in ID NO.11 and SEQ ID NO.12) were respectively inoculated in 10 mL of LB medium containing kanamycin, shaken and cultured overnight at 37° C., and transferred to 100 mL of LB medium the next day at an inoculum size of 0.5%. After culturing at 37°C for 24 hours, the fermentation broth was centrifuged at 4°C and 10,000 r/min for 10 minutes. The supernatant was the extracellular crude enzyme solution, and the supernatant from the broken cells was the intracellular crude enzyme solution, which was used for the determination of enzyme activity.

(2) Determination of the enzyme activity of L-asparaginase.

Reaction system: 100 μL of appropriately diluted enzyme solution, 800 μL of 25 mmol·L ^-1 L-asparagine solution (dissolve L-asparagine with 50 mmol·L ^-1 , pH 8 Tris-HCl buffer), in a water bath at 40 °C After reacting for 15 minutes, 100 μL of trichloroacetic acid solution (TCA) with a mass volume percent concentration of 15% (w/v) was added to terminate the reaction. In the control group, 100 μL of TCA with a concentration of 15% by mass and volume was added before the enzymatic reaction, that is, the water bath, to terminate the enzymatic reaction in advance. After the reaction, centrifuge at 10000g at room temperature for 10min, and the color reaction system is: 200μL centrifuge supernatant, 4.8mL ddH ₂ O, 200μL Nessler reagent, mix well, let stand at room temperature for 10-15min, and read at 450nm wavelength Absorbance. under the same conditions. Use different concentrations of ammonium chloride for color reaction, and draw the ammonia concentration standard curve. The enzymatic activity of L-asparaginase was calculated by measuring the amount of ammonia generated by the enzymatic reaction.

Enzyme activity unit: Under certain conditions, the amount of enzyme required to produce 1 μmol of ammonia per minute is 1 enzyme activity unit.

Under the mediation of the expression vector pMA5-SPP, the intracellular and extracellular enzyme activities of L-asparaginase were 105.4U/mL and 40.12U/mL respectively, and the enzyme protein secretion accounted for 38% of the total expression, and the extracellular secretion It is 2.95 times that mediated by the common signal peptide SP _pel .

Comparative example 1

The gene encoding the signal peptide SPphoD and the gene encoding the molecular chaperone DnaK (the amino acid sequence is shown in SEQ ID NO.17) were connected to the vector pMA5-P43 to obtain the expression vector pMA5-SPD. Replace pMA5-SPP in the embodiment with pMA5-SPD, and the rest are the same as in the embodiment. The results showed that when pMA5-SPD was used as the expression vector, the extracellular enzyme activity of L-asparaginase after expression was not significantly different from that expressed by common SPpel-mediated L-asparaginase expression strains difference.

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore The scope of protection of the present invention should be defined by the claims.

SEQUENCE LISTING

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195 200 205

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210 215 220

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Ala Lys Phe Glu Glu Leu Ser Ser His Leu Val Glu Arg Thr Met Gly

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

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

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Glu Ile Glu Arg Met Val Lys Glu Ala Glu Glu Asn Ala Asp Ala Asp

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Ala Lys Lys Lys Glu Glu Ile Glu Val Arg Asn Glu Ala Asp Gln Leu

500 505 510

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

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Glu Glu Gln Val Lys Lys Ala Asn Asp Ala Lys Asp Ala Leu Lys Ala

530 535 540

Ala Ile Glu Lys Asn Glu Phe Glu Glu Ile Lys Ala Lys Lys Asp Glu

545 550 555 560

Leu Gln Thr Ile Val Gln Glu Leu Ser Met Lys Leu Tyr Glu Glu Ala

565 570 575

Ala Lys Ala Gln Gln Ala Gln Gly Gly Ala Asn Ala Glu Gly Lys Ala

580 585 590

Asp Asp Asn Val Val Asp Ala Glu Tyr Glu Glu Val Asn Asp Asp Gln

595 600 605

Asn Lys Lys

610

Claims (10)

1. An expression vector is characterized in that a gene coding a signal peptide SPphoD and a gene coding a molecular chaperone PrsA are connected to a vector pMA5-P43 to obtain an expression vector pMA 5-SPP; the amino acid sequence of the signal peptide SPphoD is shown as SEQ ID NO.1, and the amino acid sequence of the molecular chaperone PrsA is shown as SEQ ID NO. 3.

2. The expression vector of claim 1, wherein the gene encoding the signal peptide SPphoD is ligated into the vector pMA5-P43 between the multiple cloning sites EcoR V and Kpn I, and expressed under the mediation of the P43 promoter.

3. The expression vector of claim 1, wherein the gene encoding chaperone PrsA is ligated into the vector pMA5-P43 between the multiple cloning sites BamH I and Mlu I, and expressed under the mediation of the PpHpaII promoter.

4. The expression vector of any one of claims 1 to 3, wherein the nucleotide sequence of the gene encoding the signal peptide SPphoD is shown in SEQ ID No.2, and the nucleotide sequence of the gene encoding the chaperone PrsA is shown in SEQ ID No. 4.

5. A genetically engineered bacterium characterized in that the expression vector according to any one of claims 1 to 4 is used as an expression vector.

6. The genetically engineered bacterium of claim 5, wherein the genetically engineered bacterium is hosted in Bacillus subtilis 168.

7. The genetically engineered bacterium of claim 5, wherein the genetically engineered bacterium expresses an L-asparaginase whose amino acid sequence is shown as SEQ ID No. 13.

8. The genetically engineered bacterium of claim 6, which is prepared by ligating a gene encoding L-asparaginase between restriction sites Kpn I and Hind III of pMA5-SPP to give a recombinant plasmid, and transforming the recombinant plasmid into Bacillus subtilis 168.

9. A method for preparing L-asparaginase, which is characterized in that the genetic engineering bacteria of claim 7 or 8 are inoculated in an LB culture medium and cultured for 8-12h at 35-39 ℃ and 220rpm which is 200-; inoculating to new LB culture medium in an amount of 0.5-5%, and culturing at 35-39 deg.C for 20-30 h.

10. Use of the expression vector of claims 1-4 for the production of an endogenous or exogenous protein.