CN110607319B

CN110607319B - Expression vector suitable for bacillus subtilis secretion expression protein and application

Info

Publication number: CN110607319B
Application number: CN201911040157.6A
Authority: CN
Inventors: 饶志明; 李谞; 张显; 朱曼迟; 杨套伟; 徐美娟; 邵明龙; 杜宇轩; 贾以泽; 王嘉轩
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2019-10-29
Filing date: 2019-10-29
Publication date: 2021-11-23
Anticipated expiration: 2039-10-29
Also published as: CN110607319A

Abstract

The invention discloses an expression vector suitable for bacillus subtilis to secrete expression protein and application thereof, belonging to the field of genetic engineering and biotechnology. The secretion carrier is a polypeptide which encodes a signal peptide SP_phoDThe gene (the nucleotide sequence is shown as SEQ ID NO.1) and the gene (the nucleotide sequence is shown as SEQ ID NO.3) for coding the molecular chaperone PrsA are respectively connected to pMA5-P43 to construct a secretion plasmid pMA 5-SPP. pMA5-SPP is used as a vector to express L-asparaginase in bacillus subtilis, the extracellular secretion amount accounts for 38 percent of the total expression amount, and the extracellular secretion amount is common signal peptide SP_pel2.95 times of the amount secreted under mediation.

Description

Expression vector suitable for bacillus subtilis secretion expression protein and application

Technical Field

The invention relates to an expression vector suitable for bacillus subtilis to secrete expression protein and application thereof, belonging to the field of genetic engineering and biotechnology.

Background

In the process of expressing recombinant protein by using microorganisms, good secretion performance reduces the recovery cost of products, and is an important consideration factor for whether the recombinant protein can be industrially produced. The signal peptide is a peptide chain at the N end of the secretory protein and can mediate the protein to be secreted to the outside of cells, and has important significance on the secretory expression of the protein. Molecular chaperones are capable of recognizing and binding to incompletely folded or assembled proteins in cells, and help these proteins to fold and secrete (Molecular microbiology,2010,8(4): 727-37; Microbial cell factors, 2015,14, 92). Therefore, in constructing a secretion expression vector of the protein, selecting a proper signal peptide and a proper molecular chaperone has important significance.

Bacillus subtilis has good secretion capacity due to lack of cell outer membrane, and is an ideal host for secreting biological products. Meanwhile, the Bacillus subtilis also has the characteristics of food safety, clear and good production technology and fermentation foundation of genetic information and the like, so the Bacillus subtilis is widely applied to the biological fermentation preparation of proteins, food additives and antibiotics (Trends in biological technology,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 protein by taking bacillus subtilis as host bacteria, the construction of a carrier containing effective signal peptide and molecular chaperone has important significance for protein secretion. At present, no signal peptide SP is constructed on a carrier at the same time_phoDGenes and chaperone PrsA genes to increase secretion levels of Bacillus subtilis proteins are reported in the literature.

There are relatively few reports on extracellular secretion Of L-asparaginase, And studies show that B.subtilis B11-06L-asparaginase is expressed by using Bacillus subtilis 168 as a host bacterium, And that extracellular enzyme activity accounts for 57.1% Of total enzyme activity (Journal Of Agricultural And Food Chemistry,2013,61(39):9428-9434), but recombinase activity is only 9.98U/mL. Bacillus subtilis 168 is used as a host bacterium to express Pyrococcus yayanosii-derived L-asparaginase, the extracellular and intracellular activities of which are respectively 23.31U/mL and 65.72U/mL (Scientific Reports,2018,8(1):7915), and the enzyme activity level is limited.

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

Disclosure of Invention

The first purpose of the invention is to provide a bacillus subtilis expression vector, which is obtained by connecting a gene coding a signal peptide SPphoD and a gene coding a molecular chaperone PrsA 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. In one embodiment of the invention, the gene encoding the signal peptide SPphoD is ligated into the vector pMA5-P43 between the multiple cloning sites EcoR V and Kpn I, expressed under the mediation of the P43 promoter, and the gene encoding the chaperone PrsA is ligated into the vector pMA5-P43 between the multiple cloning sites BamH I and Mlu I, expressed under the mediation of the PpHpaII promoter.

The pMA5-P43 plasmid is obtained by connecting a promoter P43 to a pMA5 vector by using enzyme digestion connection.

The construction method of the pMA5-P43 plasmid is characterized in that a forward primer F (nucleotide sequence is shown as SEQ ID NO. 15) and a reverse primer R (nucleotide sequence is shown as SEQ ID NO. 16) are used for amplifying a promoter P43 from a Bacillus subtilis 168 genome; the vector pMA5 was double digested with restriction enzymes EcoR I and Hind III; using a homologous recombination kit (

MultiS One-Step Cloning Kit, Novozam) ligated promoter P43 between the multiple Cloning sites EcoR I and Hind III of pMA5 to give recombinant plasmid pMA 5-P43.

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

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

The second purpose of the invention is to provide a genetically engineered bacterium, which takes the expression vector as an expression vector.

In one embodiment of the present invention, the genetically engineered bacterium is a bacillus subtilis host.

In one embodiment of the invention, the genetically engineered bacteria express an L-asparaginase whose amino acid sequence is shown in SEQ ID No. 13.

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

The third purpose of the invention is to provide the preparation method of the gene engineering bacteria, which is to connect the gene coding the L-asparaginase between the restriction sites Kpn I and Hind III of pMA5-SPP to obtain a recombinant plasmid, and transform the recombinant plasmid into the bacillus subtilis 168 to obtain the bacillus subtilis gene engineering bacteria.

The fourth purpose of the invention is to provide a method for preparing L-asparaginase, which comprises the steps of inoculating the genetic engineering bacteria into an LB culture medium, culturing at 35-39 ℃, 200-rpm and 220-12 h, transferring into a new LB culture medium according to 0.5-5% of the inoculum size, culturing at 35-39 ℃ for 20-30h, centrifuging the fermentation liquor, taking the supernatant as extracellular crude enzyme liquid, and taking the cell disruption supernatant as intracellular crude enzyme liquid.

The fifth purpose of the invention is to provide the application of the bacillus subtilis expression vector in preparing endogenous protein or exogenous protein.

The signal peptide SPphoD is used for mediating protein secretion, the molecular chaperone PrsA is co-expressed for assisting protein to be transported out of a cell membrane, the extracellular secretion amount of the L-asparaginase is 38% of the total expression amount, and compared with the secretion expression mediated by the common signal peptide SPpel, the extracellular secretion amount of the L-asparaginase is improved by 2.95 times by the method provided by the invention.

Detailed Description

EXAMPLE 1 construction of the expression vector pMA5-SPP

(1)SP_phoDAnd amplification of PrsA gene: respectively cloning SP (Bacillus subtilis)168 genome as a template and F1 primer (nucleotide sequence is shown in SEQ ID NO. 5) and R1 primer (nucleotide sequence is shown in SEQ ID NO. 6), F2 primer (nucleotide sequence is shown in SEQ ID NO. 7) and R2 primer (nucleotide sequence is shown in SEQ ID NO. 8) as primers_phoDAnd a PrsA gene.

(2) In the vector pMA5-P₄₃Based on (1), using a homologous recombination kit (

MultiS One-Step Cloning Kit, nuozan) will signal peptide SP_phoDThe gene is connected between the multiple cloning sites EcoR V and Kpn I, and the molecular chaperone PrsA gene is connected between the multiple cloning sites BamH I and Mlu I, so as to construct an expression vector pMA5-SPP beneficial to protein secretion.

EXAMPLE 2 construction of L-asparaginase secretion-expressing Strain

The plasmid pMA 5-pyranase (the construction method is shown in the literature: Xu L, Xian Z, Shuqin X, et al]Scientific Reports,2018,8(1):7915.) as a template, and F3 primer (nucleotide sequence shown in SEQ ID NO. 9) and R4 primer (nucleotide sequence shown in SEQ ID NO. 10) as primers, a gene (nucleotide sequence shown in SEQ ID NO. 14) encoding L-asparaginase was amplified, and a homologous recombination kit was used (

MultiS One-Step Cloning Kit) was ligated between restriction sites Kpn I and Hind III of pMA5-SPP to obtain a recombinant plasmid. The obtained recombinant plasmid is transformed into bacillus subtilis 168 competent cells by a chemical method, and an L-asparaginase secretion expression strain B.subtilis/pMA5-SPP-pyasnase is constructed

Example 3B. subtilis/pMA5-SPP-pyasnase secretion assay

(1) The recombinant strain B.subtilis/pMA5-SPP-pyasnase obtained in example 2 and an L-asparaginase expression strain (the construction method is the same as in examples 1 and 2, and the primer sequences are shown as SEQ ID NO.11 and SEQ ID NO. 12) only mediated by common SPpel are respectively inoculated into 10mL LB culture medium containing kanamycin, shaking culture is carried out at 37 ℃ for overnight, the next day is inoculated into 100mL LB culture medium according to the inoculum size of 0.5 percent, culture is carried out at 37 ℃ for 24h, fermentation liquor is taken and centrifuged at 10000r/min for 10min at 4 ℃, the supernatant is extracellular crude enzyme liquid, and the cell crushing supernatant is intracellular crude enzyme liquid and is used for measuring the enzyme activity.

(2) And (3) measuring the enzyme activity of the L-asparaginase.

Reaction system: 100. mu.L of the enzyme solution diluted as appropriate, 800. mu.L of 25 mmol. multidot.L^-1L-asparagine solution (with50mmol·L^-1Dissolving L-asparagine in Tris-HCl buffer solution with pH 8), reacting in water bath at 40 ℃ for 15min, and adding 100 mu L of trichloroacetic acid solution (TCA) with the mass volume percentage concentration of 15% (w/v) to terminate the reaction. In the control group, 100. mu.L of TCA having a mass-volume percent concentration of 15% was added before the enzyme reaction, i.e., the water bath, to terminate the enzyme reaction early. Centrifuging at normal temperature for 10min at 10000g of rotating speed after reaction, wherein the color reaction system is as follows: 200 μ L of the centrifuged supernatant, 4.8mL of ddH₂O and 200 mul of Neusler reagent, standing for 10-15min at room temperature after uniformly mixing, and reading the absorbance at the wavelength of 450 nm. Under the same conditions. And performing color reaction by using ammonium chloride with different concentrations, and drawing an ammonia concentration standard curve. The L-asparaginase enzyme activity was calculated by measuring the amount of ammonia produced by the enzymatic reaction.

The enzyme activity unit is as follows: under certain conditions, the enzyme amount required for generating 1 mu mol of ammonia gas per minute is 1 enzyme activity unit.

Under the mediation of the expression vector pMA5-SPP, the intracellular and extracellular enzyme activities of the L-asparaginase are respectively 105.4U/mL and 40.12U/mL, the secretion amount of the enzyme protein accounts for 38 percent of the total expression amount, and the extracellular secretion amount is the common signal peptide SP_pel2.95 fold under mediation.

Comparative example 1

The gene coding the signal peptide SPphoD and the gene coding the molecular chaperone DnaK (the amino acid sequence is shown in SEQ ID NO. 17) are connected to a vector pMA5-P43 to obtain an expression vector pMA 5-SPD. The pMA5-SPP in the example was replaced with pMA5-SPD, the remainder being identical to the examples. The result shows that the expressed L-asparaginase extracellular enzyme activity has no significant difference compared with the extracellular enzyme activity expressed by the common SPpel-mediated L-asparaginase expression strain by taking pMA5-SPD as an expression vector.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

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

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

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

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Asn Lys Lys

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Claims

1. A method for preparing L-asparaginase is characterized in that genetically engineered bacteria are inoculated in an LB culture medium and cultured for 8-12h at 35-39 ℃ and 200-220 rpm; inoculating into new LB culture medium in an amount of 0.5-5%, and culturing at 35-39 deg.C for 20-30 hr;

the genetic engineering bacteria take Bacillus subtilis 168 as a host and carry an expression vector pMA5-SPP,

the construction method of the expression vector pMA5-SPP comprises the following steps: connecting a gene encoding a signal peptide SPphoD and a gene encoding a molecular chaperone PrsA 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;

the gene coding the signal peptide SPphoD is connected between the multiple cloning sites EcoR V and Kpn I of the vector pMA5-P43 and is expressed under the mediation of a P43 promoter;

the coding molecular chaperone PrsA gene is connected between the multiple cloning sites BamH I and Mlu I of the vector pMA5-P43 and is expressed under the mediation of a PpHpaII promoter;

the nucleotide sequence of the gene for coding the signal peptide SPphoD is shown as SEQ ID NO.2, and the nucleotide sequence of the gene for coding the molecular chaperone PrsA is shown as SEQ ID NO. 4;

the amino acid sequence of the L-asparaginase is shown as SEQ ID NO. 13.