CN112195192A - Method for effectively expressing NaD1 protein by using yeast expression system - Google Patents
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Abstract
The invention discloses a method for effectively expressing NaD1 protein by using a yeast expression system. The method constructs a recombinant plasmid pPIC9K-NaD1 between EcoR I and Not I which are connected to a pPIC9K vector by double enzyme digestion of a flower tobacco NaD1 gene and obtains a recombinant NaD1 protein expression strain which is transferred into GS115 to obtain the recombinant NaD1 protein expression strain, the strain can effectively, stably and massively express the recombinant NaD1 protein, the total amount of the purified recombinant protein is 1mg, and the protein purity reaches 85%. The product is detected by Western Blot and SDS-PAGE, the molecular weight of the actual recombinant protein is about 20kDa, the theoretical size is about 11kDa, and the protein is presumed to be possibly glycosylated modified and exist in a polymer form in Pichia pastoris. According to the invention, the NaD1 protein is successfully expressed through a yeast expression system, and an experimental basis is provided for further research on an action mechanism of the NaD1 protein.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a method for effectively expressing NaD1 protein by using a yeast expression system.
Background
At present, the production of medicinal proteins by cell engineering has become one of three major trends of biotechnology pharmacy in the world. An important scientific research technology in a foreign protein expression system is Pichia pastoris (Pichia pastoris), the time for using the technology in the expression of the foreign protein can be traced back to the present in 1987, and researches show that more than 500 proteins from various aspects realize the secretion expression of the Pichia pastoris, wherein medicinal proteins which are put into industrialization occupy a very important part. Pichia pastoris has the characteristics of easy mass propagation and culture of prokaryotes and the like, and has the capability of correctly folding a gene product and modifying and processing after translation by eukaryotes.
The Nicotiana alata defensin 1(NaD1) protein is a plant defensin from ornamental floral tobacco (Nicotiana alata). According to previous studies, the mechanism by which the NaD1 protein kills fungal cells in Nicotiana alata is the specific interaction of the fungal cell wall before passing through the plasma membrane and entering the fungal cytoplasm. Upon entering the cell, NaD1 rapidly triggers the production of Reactive Oxygen Species (ROS), permeabilization of the fungal plasma membrane, and cell death. Therefore, it has good antibacterial activity against various pathogenic fungi such as Fusarium oxysporum (Fusarium oxysporum), Botrytis cinerea (Botrytis cinerea), Candida (Candida albicans) and the like. It has also been shown that the NaD1 protein can mediate activity against tumor cell lines through specific and high affinity interactions with phosphoinositide 4, 5-diphosphate, but the detailed mechanism is still under further investigation.
Disclosure of Invention
The invention aims to provide a pichia pastoris engineering bacterium for secretory expression of a flower tobacco NaD1 gene and a construction method thereof, which can efficiently and safely carry out extracellular secretory expression of the flower tobacco NaD1 gene.
The technical scheme for realizing the purpose of the invention is as follows:
the NaD1 gene vector pPIC9K-NaD1 of the nicotiana japonica has a base sequence shown as SEQ ID No. 1.
The engineering bacteria of flower tobacco NaD1 gene, whose receptor bacteria is Pichia pastoris GS115, is transferred into the above-mentioned vector pPIC9K-NaD 1.
A method for effectively expressing NaD1 protein by using a yeast expression system comprises the following steps:
(1) the NaD1 gene of the nicotiana tabacum is subjected to double digestion and is connected to EcoR I and Not I of a pPIC9K vector to construct a recombinant plasmid pPIC9K-NaD1, and the nucleotide sequence of the recombinant plasmid is shown as SEQ ID No. 1;
(2) carrying out agarose gel electrophoresis on the plasmid after linearization by Sac I endonuclease, and recovering the digestion product; electrically converting and recovering the product in GS115 competent cells to obtain recombinant positive clone;
(3) inoculating the recombinant positive clone into a BMGY liquid culture medium, culturing at 30 ℃ and 200rpm, centrifuging to remove liquid culture medium supernatant, inoculating into a BMMY culture medium, adding a defoaming agent, culturing at 30 ℃ and 200rpm, and after the culture is finished; then 10mL of methanol is added for induction, the culture is carried out at 30 ℃ and 200rpm, the centrifugation is carried out at 8000rpm, and the supernatant fluid is taken, wherein the supernatant fluid contains soluble NaD1 protein.
Culturing in BMGY liquid culture medium for 24 hr, adding defoaming agent to BMMY culture medium, culturing for 24 hr, adding methanol for induction, and culturing for 24 hr.
The step (3) is followed by a purification step: A. adjusting the concentrated supernatant to pH8.0 with 1M Tris liquid at pH 9.0; filtering with a filter with the filtering specification of 0.22 mu m to obtain a supernatant protein solution, loading the supernatant protein solution to a prepared nickel column, washing the nickel column with NTA-0 buffer solution with the pH value of 8.0 until the effluent liquid does not contain protein, eluting with an imidazole solution, collecting the eluent of a section showing blue color in G250 detection, and performing ultrafiltration concentration by an ultrafiltration tube to obtain the soluble NaD1 protein.
The concentration of the imidazole solution is 250mM, and the specification of an ultrafiltration tube is 10 kDa.
The method comprises the steps of connecting the NaD1 gene of the flower tobacco to EcoR I and Not I of a pPIC9K vector through double enzyme digestion to construct a recombinant plasmid pPIC9K-NaD1 (partial sequences of the NaD1 gene of the flower tobacco are shown as SEQ ID No.2 and SEQ ID No. 3), then transforming the gene into an E.coli TOP10 strain, coating the strain on an LB plate (containing kanamycin and ampicillin resistance), selecting a single colony successfully screened by using an aseptic gun head, transferring the single colony into an LB liquid culture medium, taking a bacterial liquid for seed preservation, and inoculating the residual bacterial liquid into the LB liquid culture medium (containing ampicillin and kanamycin resistance) for culture. Collecting thalli and then carrying out mass extraction of plasmids; carrying out agarose gel electrophoresis on the recombinant plasmid pPIC9K-NaD1 identified by electrophoresis after enzyme digestion after linearization by Sac I endonuclease, and recovering an enzyme digestion product; and (3) electrically converting and recycling a product in GS115 competent cells, screening to obtain high-expression pichia pastoris gene engineering bacteria after pichia pastoris is converted, and obtaining NaD1 recombinant protein with the purity of 85 percent through preliminary fermentation and purification steps.
The invention uses a pichia pastoris GS115 expression system to induce and express pPIC9K-NaD1 gene recombinant vector in a secreted soluble form, and Ni-NTA purification technology to extract recombinant NaD1 protein, thereby providing a good foundation for further researching the mechanism of killing fungi by the NaD1 protein and the anticancer function.
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FIG. 1 is an agarose gel electrophoresis of the plasmid and the cleavage products.
In the figure, M is Marker (10000/8000/6000/5000/4000/3000/2000/1500/1000/800/500/300 bp); 1: a plasmid; 2: and (3) carrying out plasmid digestion on the product.
FIG. 2 shows agarose gel electrophoresis detection of PCR products of positive clones.
In the figure, M is DL2000 Marker; negative control; positive control; 1-9, positive clone PCR product.
FIG. 3 is an SDS-PAGE electrophoresis of small expression products from positive clones.
In the figure, M is a protein molecular weight standard (116.0/66.2/45.0/35.0/25.0/18.4/14.4 kDa); small expression of the product by empty vector positive cloning; 1-10, positive clone expresses product in small quantity.
FIG. 4 is an SDS-PAGE of the Ni-NTA purified eluate protein of the supernatant.
In the figure, M is a protein molecular weight standard (116.0/66.2/45.0/35.0/25.0/18.4/14.4 kDa); 1, eluting the concentrated protein.
FIG. 5 shows WesternBlot assay of the supernatant Ni-NTA purified eluate protein.
In the figure, M is a protein molecular weight standard (250/150/100/70/50/40/35/25/20/15/10 kDa); positive control about 40 kDa; and 1-2, eluting the concentrated protein.
Detailed Description
The present invention will be described in further detail with reference to the following detailed description and accompanying drawings.
All the following biological materials are commercially available, and the laboratory of the applicant stores the biological materials and can issue the biological materials to the outside in a public way.
Example 1 construction of expression vector and Mass extraction of recombinant plasmid
The Wuhan Kingkunry company clones NaD1 gene in pPIC9K vector with EcoRI and NotI restriction enzyme, transforms the constructed recombinant plasmid pPIC9K-NaD1 into E.coli TOP10 strain and coats it on LB plate (containing kanamycin and ampicillin resistance), picks up single colony containing successfully screened colony with aseptic gun head and transfers it into 3mL LB liquid culture medium, and shake-cultivates it overnight at 37 deg.C. Then 700. mu.L of the bacterial liquid is taken for seed preservation, and the rest of the bacterial liquid is inoculated in 300mL of LB liquid culture medium (containing ampicillin and kanamycin resistance) and cultured by shaking overnight at 37 ℃. After the thalli are collected, a large amount of plasmid is extracted. The results of the plasmids identified by electrophoresis after enzyme digestion show that the purity of the extracted plasmids reaches the standard, and the sizes of the plasmids and the size of the enzyme digestion products accord with expectations, as shown in figure 1. The nucleotide sequence of the recombinant plasmid is shown as SEQ ID No. 1.
Example 2 competent electrotransformation and Positive clone screening PCR identification
Single colonies of GS115 on BMGY-streaked plates were picked and cultured at 30 ℃ for 24h at 200rpm in 15mL BMGY liquid medium. Inoculating 100 mu L of bacterial liquid into 100mL of BMGY culture medium, culturing at 30 ℃ and 200rpm until the OD600 value is 0.8-1.0, pouring the cultured bacterial liquid into a precooled centrifugal tube, centrifuging at 3000g and 4 ℃ for 5min, and removing the supernatant. The cells were resuspended in ice sterile water, centrifuged at 3000g 4 ℃ for 5min and the supernatant removed. The above operation is repeated with 1/2 volumes of ice sterile water. After ice 1M sorbitol resuspended the pellet, repeat the above procedure. Resuspend 50mL of the pellet with 200. mu.L of ice 1M sorbitol, and store the competent cells in 80. mu.L/tube in an ultra-low temperature refrigerator. Carrying out agarose gel electrophoresis on the extracted plasmid after linearization by Sac I endonuclease, and recovering the digestion product; electrically converting and recovering the product in GS115 competent cells, and setting parameters to be 1500V and 5 ms; the selection was carried out by plating the electrically transformed cells on RDB + G418 plates. The recombinant positive clone obtained by electric transformation is selected, shaken for one colony overnight, set with a blank control (without plasmid template) and a positive control (with 0.5 uL plasmid added), and is used for colony PCR detection. The PCR primers are shown in Table 1.
Wherein, the PCR reaction system is as follows: dNTP 1.6. mu.L, 10 XBuffer 2.0. mu.L, upstream and downstream primers 0.2. mu.L each, Enzyme 0.2. mu.L, ddH2O to make up the total system to 20.0. mu.L. The PCR reaction program is: 94 ℃ for 5min, (94 ℃ for 30s,54 ℃ for 30s,72 ℃ for 90s) for 30 cycles, 72 ℃ for 7min, 4 ℃ infinity.
After detecting the PCR product by 1.2% agarose gel electrophoresis, the result showed that the size of the target band obtained by PCR amplification coincided with the expected size of the target band, and the size was about 278bp, as shown in FIG. 2. The sequencing is correctly shown by the post-sequencing result of Wuhan Kingkurui bioengineering GmbH.
TABLE 1 PCR primer sequence Listing
Example 3 detection of Small expression products of Positive clones by SDS-PAGE
Selecting clones with positive colony PCR identification and preserving the clones in an ultra-low temperature refrigerator. And picking positive strains by using a gun head to cultivate the positive strains in 1.5mL BMGY liquid medium in a shaker at 30 ℃ and 200rpm for 48 h. Standing the bacterial liquid for 5-6 h, removing the supernatant, adding 1mL of BMMY, and culturing at 30 ℃ and 200rpm for 24 h; the induction conditions were 200. mu.L YP medium containing 5% methanol at 30 ℃ and 200rpm for 24 h; centrifuging at 11000rpm for 10min, taking supernatant, and adding 150 μ L TCA for ice bath for 2 h; centrifuging at 11000rpm for 10min, removing supernatant, adding 200 mu L of acetone to wash and precipitate, repeating the steps, and drying in a 60 ℃ oven for 15-20 min; after addition of 20. mu.L of 1 × loading buffer, the pellet was resuspended and solubilized and then examined by SDS-PAGE. The results showed that the expression of all the positive cloned recombinant proteins was less pronounced, as shown in FIG. 3.
Example 4 protein Mass expression and purification of recombinant protein SDS-PAGE and Western Blot detection
And single colonies with higher positive expression level are identified by SDS-PAGE, inoculated in 1L BMGY liquid medium and cultured for 24h at 30 ℃ and 200 rpm. Centrifuging for 3min at 8000rpm, removing supernatant, inoculating to 1L BMMY culture medium, adding 200 μ L defoaming agent (commercially available product "DIDEN"), and culturing at 30 deg.C under 200rpm for 24 h. 10mL of methanol was added for induction, and the cells were incubated at 30 ℃ and 200rpm for 24 hours. Repeating the above steps, centrifuging at 8000rpm for 5min, and collecting supernatant. The solution was filtered through a stainless steel disc filter and concentrated to 200mL by dialysis column for subsequent purification.
Adjusting the pH of the concentrated supernatant to 8.0 with 1M Tris (pH9.0); the filtration specification was 0.22 μm. And (3) loading the prepared nickel column with a supernatant protein solution at the flow rate of 1mL/min, washing the nickel column with NTA-0 buffer solution with pH8.0 until the effluent liquid does not contain protein, eluting with 250mM imidazole, collecting a section showing blue color in G250 detection, performing ultrafiltration concentration on the eluate to 1-2 mL through a 10kDa ultrafiltration tube, and performing SDS-PAGE detection on the concentrated solution.
Carrying out 15% SDS-PAGE electrophoresis on the purified NaD1 recombinant protein, then adjusting the voltage to 200V, and then adding 1.5mA/cm2And (3) transferring the gel volume to the membrane for 1.5h, taking out the membrane, immersing the cleaned membrane in a sealing solution, incubating for 2h at 37 ℃, and cleaning with a PBST solution. After the washing is finished, the solution is respectively incubated for 60min in the diluted primary antibody and secondary antibody, and the solution is gently rinsed for 5min in PBST solution, and the operation is repeated for 3 times. After development using the procedure of the ECL kit, scanning was performed and the record was saved. 200 mu L of the purified NaD1 recombinant protein is added with 2 Xloading buffer solution, boiled for 10 minutes and sent to Wuhan Kingkurui Co Ltd for protein mass spectrum identification.
A small amount of positive clones with the highest expression level are selected for mass culture to obtain a large amount of expression recombinant proteins, the recombinant proteins comprise His tag (-2 kDa), the molecular weight of the actual recombinant proteins is about 20kDa and the theoretical size is about 11kDa through SDS-PAGE and Western Blot detection, and protein bands are in a dispersed state and belong to the characteristic of glycosylation modification. And NaD1 protein can form polymer form to play its antibacterial function, supposedly the protein may be glycosylation modified and contain polymer form. The NaD1 protein was purified by Ni-NTA purification technique to obtain 1mg of recombinant protein, which was 85% pure, and the results are shown in FIGS. 4 and 5.
Example 5 analysis of the yield results of NaD1 recombinant protein
According to the fermentation conditions of the pichia pastoris, the pichia pastoris bacterial liquid containing pPIC9K-NaD1 is inoculated in 1L BMGY liquid culture medium for mass expression. About 0.25mg of NaD1 recombinant protein can be obtained by purifying 1L of zymocyte liquid, and the calculated protein yield is 0.25 mg/L.
Example 6 Mass Spectrometry identification of NaD1 recombinant protein
When the confidence conf is more than or equal to 95 percent and the Unique peptides are more than or equal to 1, the total number of the proteins identified by the NaD1 protein sample is respectively 8, the ratio of the highest 12 percent of Coverage is 6His-NaD1, the molecular weight is 9.7kDa, and the molecular weight is consistent with the theoretical molecular weight of NaD1 recombinant protein. The identification result shows that NaD1 recombinant protein is successfully purified from the yeast expression system, and the result is shown in Table 2.
TABLE 2 protein-related information Table
The invention uses a pichia pastoris GS115 expression system to induce and express pPIC9K-NaD1 gene recombinant vector in a secreted soluble form, and Ni-NTA purification technology to extract recombinant NaD1 protein, thereby providing a good foundation for further researching the mechanism of killing fungi by the NaD1 protein and the anticancer function.
The culture medium and the formula used in the invention are as follows:
LB liquid medium (1L, PH ═ 7.0): 950ml of deionized water, 10g of tryptone, 5g of yeast extract and 10g of NaCl
LB liquid medium (1L, PH ═ 7.0): 950ml of deionized water + tryptone 10g + Yeast extract 5g + NaCl 10g +15g agar
BMGY liquid Medium: yeast extract 1% + tryptone 2% + potassium phosphate buffer (pH6.0)100mmol/L + YNB 1.34% + Biotin (4X 10-5)% + Glycerol 1%
BMMY liquid medium: yeast extract 1% + tryptone 2% + Potassium phosphate buffer (pH6.0)100mmol/L + YNB 1.34% Biotin (4X 10-5)% + methanol 3%
1M Tris (pH9.0) in Tris, deionized water, and hydrochloric acid to adjust the pH to 9.0.
NTA-0 buffer: Tris-HCl 200mmol/L (pH 7.9) + NaCl 0.5mol/L + Glycerol 10%
PBST solution: na (Na)2HPO4(8mM)+NaCl(0.136M)+KH2PO4(2mM)+KCl(2.6mM)+Tween-20 0.05％(v/v)
The foregoing is a more detailed description of the present invention that is presented in conjunction with specific embodiments, and the practice of the invention is not to be considered limited to those descriptions. It will be apparent to those skilled in the art that a number of simple derivations or substitutions can be made without departing from the inventive concept.
Sequence listing
<110> Guizhou university
China Tobacco Corporation Guizhou Provincial Co.
<120> a method for efficiently expressing NaD1 protein using yeast expression system
<141> 2020-09-30
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 278
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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gaattcatgc accatcacca tcaccataga gaatgcaaag cagaaagcat caccttctct 60
ggattatgcg tttccaaacc accatgcaga aaagcttgta tctgtgaggg atttactaat 120
ggtcattgta gcaaaatcct cagaaggtgc ctatgcacta cgccaagtgt atttgatgag 180
aagatgatca aaacaggagc tgaaactttg ggtgaggaag caaaaacttt ggctgcaact 240
ttgcttgaag aagagataat ggataactaa gcggccgc 278
<210> 2
<211> 243
<212> DNA
<213> floral tobacco (Nicotiana alata)
<400> 2
agagaatgca aagcagaaag catcaccttc tctggattat gcgtttccaa accaccatgc 60
agaaaagctt gtatctgtga gggatttact aatggtcatt gtagcaaaat cctcagaagg 120
tgcctatgca ctacgccaag tgtatttgat gagaagatga tcaaaacagg agctgaaact 180
ttgggtgagg aagcaaaaac tttggctgca actttgcttg aagaagagat aatggataac 240
taa 243
<210> 3
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<213> floral tobacco (Nicotiana alata)
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Arg Glu Cys Lys Ala Glu Ser Ile Thr Phe Ser Gly Leu Cys Val Ser
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Lys Pro Pro Cys Arg Lys Ala Cys Ile Cys Glu Gly Phe Thr Asn Gly
20 25 30
His Cys Ser Lys Ile Leu Arg Arg Cys Leu Cys Thr Thr Pro Ser Val
35 40 45
Phe Asp Glu Lys Met Ile Lys Thr Gly Ala Glu Thr Leu Gly Glu Glu
50 55 60
Ala Lys Thr Leu Ala Ala Thr Leu Leu Glu Glu Glu Ile Met Asp Asn
65 70 75 80
Claims (6)
1. The NaD1 gene of nicotiana florida constructs a vector pPIC9K-NaD1, and the base sequence is shown as SEQ No. 1.
2. The engineering bacteria of flower tobacco NaD1 gene, whose receptor bacteria is Pichia pastoris GS115, is transferred with the vector pPIC9K-NaD1 of claim 1.
3. A method for effectively expressing NaD1 protein by using a yeast expression system comprises the following steps:
(1) the NaD1 gene of the nicotiana tabacum is subjected to double digestion and is connected to EcoR I and Not I of a pPIC9K vector to construct a recombinant plasmid pPIC9K-NaD1, and the nucleotide sequence of the recombinant plasmid is shown as SEQ ID No. 1;
(2) carrying out agarose gel electrophoresis on the plasmid after linearization by Sac I endonuclease, and recovering the digestion product; electrically converting and recovering the product in GS115 competent cells to obtain recombinant positive clone;
(3) inoculating the recombinant positive clone into a BMGY liquid culture medium, culturing at 30 ℃ and 200rpm, centrifuging to remove the supernatant of the liquid culture medium, inoculating into a BMMY culture medium, adding an antifoaming agent, culturing at 30 ℃ and 200rpm, adding 10mL of methanol for induction after the culture is finished, culturing at 30 ℃ and 200rpm, centrifuging at 8000rpm, and taking the supernatant, wherein the supernatant contains soluble NaD1 protein.
4. The method of claim 3, wherein the BMGY liquid medium is cultured for 24 hours, wherein the BMMY medium is cultured for 24 hours with the addition of a foam killer for 24 hours, then is induced by the addition of methanol, and is cultured for 24 hours.
5. The method of claim 4, further comprising a purification step after step (3): A. adjusting the concentrated supernatant to pH8.0 with 1M Tris liquid at pH 9.0; filtering with a filter with the filtering specification of 0.22 mu m to obtain a supernatant protein solution, loading the supernatant protein solution to a prepared nickel column, washing the nickel column with NTA-0 buffer solution with the pH value of 8.0 until the effluent liquid does not contain protein, eluting with an imidazole solution, collecting the eluent of a section showing blue color in G250 detection, and performing ultrafiltration concentration by an ultrafiltration tube to obtain the soluble NaD1 protein.
6. The method of claim 5, wherein the imidazole solution has a concentration of 250mM and an ultrafiltration tube size of 10 kDa.
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| CN110157710A (en) * | 2019-07-11 | 2019-08-23 | 贵州大学 | Henbane NaD1 gene promoter and application thereof |
| CN110157710B (en) * | 2019-07-11 | 2022-12-27 | 贵州大学 | NaD1 gene promoter of nicotiana tabacum and application thereof |
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Application publication date: 20210108 |