CN108034670B - Column-free low-cost preparation method of food-derived soybean bioactive peptide Lunasin - Google Patents
Column-free low-cost preparation method of food-derived soybean bioactive peptide Lunasin Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and relates to a column-free low-cost preparation method of a food-derived soybean bioactive peptide Lunasin. The method comprises the steps of firstly, obtaining a Lunasin gene fragment by adopting an overlappingextension PCR method, obtaining an NpuC-Lunasin fusion fragment on the basis of the Lunasin gene fragment, and realizing the fusion expression of the fusion fragment in an expression vector pET 26; fusing and expressing an ELP label of the elastin-like protein and the N end of a DnaE split intein; finally, the recombinant Lunasin polypeptide of the natural N end is separated and purified simply, rapidly and in a column-free manner at low cost by utilizing the trans-splicing of the broken intein and the reversible cycle phase change of the ELP, thereby laying a foundation for the development and utilization of the Lunasin in the fields of functional foods, health care products, biological medicines and the like.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a column-free low-cost preparation method of a food-derived soybean bioactive peptide Lunasin.
Background
Lunasin (Lunasin) is a bioactive peptide of 43 amino acids derived from soybean, and was first isolated and identified from soybean seeds in 1987 by a member of the institute of medicine, university of new diarrhoea, japan. In recent years, studies have shown that the active peptide has the effect of inhibiting cell proliferation. Lunasin contains a cell adhesion sequence Arg-Gly-Asp (RGD), which can be connected with extracellular matrix to cause the Lunasin to be internalized into nucleus, and the C terminal of the Lunasin contains 8 aspartic acid residues which can be combined with deacetylated chromatin region (such as centromere), so that the centromere complex can not be formed correctly, and microtubules can not capture the centromere, thereby causing mitosis to be stopped and inhibiting the occurrence of mitosis. In addition, recent studies have shown that Lunasin also inhibits the occurrence of inflammation by inhibiting the NF-kB signaling pathway. Therefore, the food-derived soybean bioactive peptide Lunasin can not only provide protein nutrition required by growth and development for organisms, but also have good development prospects in the fields of functional foods, health-care products, biological medicines and the like in the future due to potential biological activities of cell proliferation inhibition, cancer resistance, anti-inflammation and the like.
However, at present, Lunasin is usually obtained from soybeans by separation means such as chromatography, and the method has the disadvantages of complex operation, complex steps, poor repeatability, high cost and no contribution to industrial production. The other method is chemical synthesis, which increases the purity of the product, but has high cost and is only suitable for laboratory-based research. In addition, the method for preparing Lunasin by adopting a genetic engineering means can obtain a large amount of recombinant Lunasin protein in escherichia coli, but an affinity chromatography method is used in the purification stage, and the obtained recombinant protein often has a purification tag and is a non-natural N-terminal protein, which often influences the biological activity and the immune response of the recombinant Lunasin protein. The natural N-terminal Lunasin obtained by exploring a simple, quick and low-cost method has important significance for development and utilization of the natural N-terminal Lunasin.
Disclosure of Invention
The invention aims to establish a whole set of Lunasin prokaryotic recombinant expression and purification system, and provides a column-free low-cost preparation method of food-borne soybean bioactive peptide Lunasin by adopting a column-free purification system.
In order to realize the purpose, the invention adopts the following technical scheme:
a non-column low-cost preparation method of food-derived soybean bioactive peptide Lunasin comprises the following steps: firstly, acquiring a Lunasin gene fragment by adopting an Overlapping Extension PCR method, and acquiring an NpuC-Lunasin fusion fragment on the basis of the Lunasin gene fragment, so as to realize the fusion expression of the fusion fragment in an expression vector pET 26; fusing and expressing an ELP label of the elastin-like protein and the N end of a DnaE split intein; finally, the natural N-terminal recombinant Lunasin polypeptide is separated and purified simply, quickly and inexpensively in a column-free manner by utilizing trans-splicing of the broken intein and reversible cycle phase change of the ELP.
The preparation method comprises the following specific steps:
obtaining the lunasin Gene
The upstream and downstream primers are as follows:
an upstream primer:
5-TCTAAATGGCAGCACCAGCAGGACTCTTGCCGTAAACAGCTGCAGGGTGTTAACCTG ACCCCGTGCGAAAAACACATC-3
a downstream primer:
5-TTACTAGTCATCGTCATCGTCATCGTCATCGTCACCACGACCCTGGATTTTTTCCATGA TGTGTTTTTCGCACGGGGTC-3
(1) the Lunasin-encoding Gene was codon-optimized and synthesized using OPTIMIZER and Gene Designer according to Lunasin amino acid sequence published on PDB, and delivered to shanghai bio corporation for synthesis. And performing overlapping extension PCR on the upstream primer and the downstream primer to obtain lunasin. And the obtained PCR product is identified by 2% lipotrope gel electrophoresis, and then lunasin is recovered by a gel recovery kit.
(2) Construction of Lunasin gene T vector
The lunasin gene is connected to pMD19-T simple vector, then transformed into DH5 competent bacteria, spread and contain resistant LB plate, obtained colony is identified by PCR, and the obtained positive clone is sent to Shanghai bioengineering limited company for sequencing after amplification culture. The sequencing result is compared with the Lunasin sequence provided by GenBank, and the recombinant plasmid is named as pMD19-T-Lunasin after the sequence is identified to be correct.
Construction and identification of NpuC-Lunasin fusion gene cloning vector
(1) Lunasin gene amplification
Designing a primer:
an upstream primer 1: TAGCTTCTAATTCTAAATGGCAGCACCAGCAG
A downstream primer 1: CCGCTCGAGTTACTAGTCATCGTCATCGTCATCG
Using T vector pMD19-T-Lunasin containing Lunasin fragment as template, upstream primer 1 and downstream primer 1 were used to perform PCR amplification on lunsian gene.
(2) NpuC gene amplification
Designing a primer:
an upstream primer 2: TTAGAAGGCATATGATCAAAATAGCCACACGTAAATATTTAGG
A downstream primer 2: GCTGGTGCTGCCATTTAGAATTAGAAGCTATGAAGCCATTTTTG
The NpuC gene was RCR amplified using pET-NpuC-GFP plasmid as a template and the forward primer 2 and the reverse primer 2.
(3) NpuC-Lunasin fusion gene amplification
Respectively using the NpuC and Lunasin templates, using an upstream primer 2 and a downstream primer 1 to perform overlap, using the result as a template to perform PCR amplification, and recovering the NpuC-Lunasin gene from a gel recovery kit after identifying the PCR product by 2% agarose gel electrophoresis.
(4) TA cloning of fusion-based NpuC-Lunasin fragment
The gene NpuC-Lunasin is connected to pMD19-T simple vector, then transformed into DH5 competent bacteria, spread and contain resistant LB plate, the obtained colony is identified by PCR, and the positive clone is selected, amplified and cultured and sent to Shanghai bioengineering limited company for sequencing. And (3) analyzing the sequencing result by using Sequence software, comparing the sequencing result with a known Sequence, identifying the Sequence to be correct, and then naming the recombinant plasmid as PMD 19-T-NpuC-Lunasin.
3. Construction and identification of fusion gene NpuC-Lunasin expression vector
Coli containing plasmids PMD19-T-NpuC-Lunasin and pET26-b (+) were separately grown and plasmid-extracted, followed by double digestion with Nde I and Xho I. And cutting the gel, recovering the NpuC-Lunasin fragment and the pET26-b (+) target fragment of the enzyme digestion product, and connecting overnight. The ligation products were transformed into E.coli DH5 competent cells and plated on resistant LB solid medium. After culturing for 16h, selecting positive colonies for amplification culture, carrying out plasmid extraction and Nde I and Xho I double enzyme digestion identification, and naming the successfully constructed recombinant expression plasmid as pET 26-NpuC-Lunasin.
Inducible expression and purification of lunasin in Escherichia coli
(1) Inducible expression of pET26-NpuC-Lunasin polypeptide
pET-26-NpuC-Lunasin plasmid is extracted from the screened positive strain, and expression strain BL21(DE3) competent cells prepared by transforming calcium chloride with 1 mu L of plasmid are taken. Spread on LB plate and cultured at 37 ℃ in an inverted manner. And inoculating the positive strain into an LB culture medium, culturing at 37 ℃, and carrying out IPTG (isopropyl-beta-thiogalactoside) induced expression for 6 h. The bacterial solution was centrifuged at 6000rpm, and the supernatant was discarded. 10mL of 20mM Tris-HCl 7.4buffer (containing PMSF) is added into each gram of wet bacteria, and the wet bacteria are subjected to ultrasonic disruption. The disrupted cells were identified by SDS-PAGE.
(2) ELP mediated Lunasin separation and purification
1) ElP-N inducible expression and purification
The strain containing pET-ELP-N is inoculated into LB culture medium for culture at 37 ℃, and IPTG induced expression is carried out for 6 h. The bacterial solution was centrifuged at 6000rpm, and the supernatant was discarded. 10mL of 20mM Tris-HCl 7.4buffer (containing PMSF) is added into each gram of wet bacteria, and the wet bacteria are subjected to ultrasonic disruption. And (3) mixing the centrifugal supernatant of the recombinant bacterium lysate with 0.8mol/L (NH4)2S04 with the same volume, incubating for 10min at 30 ℃, centrifuging for 5min at 14000g, and collecting the centrifugal precipitate to obtain the primarily purified ELP-N fusion protein.
2) Lunasin purification
Mixing pre-purified ELP-N and NpuC-Lunasin ultrasonic supernatant, incubating for 10min at room temperature, mixing the supernatant mixed liquor with equal volume of 0.8mol/L (NH4)2S04, incubating for 10min at determined 30 ℃, 14000g centrifuging for 5min, collecting the centrifugal precipitate in optimized cutting fluid (50mM DTT), incubating for 3h at room temperature, adding equal volume of 0.8mol/L (NH4)2S04, incubating for 10min, 14000g centrifuging for 5min, sampling the supernatant and the precipitate, and carrying out TiCine/SDS-PAGE analysis.
Compared with the prior art, the invention has the beneficial effects that: constructing an expression and purification system of the N & ltu & gt C-Lunasin recombinant protein mediated by the broken intein and the elastin-like protein (ELP), firstly synthesizing a Lunasin gene, secondly obtaining a fusion gene N & ltu & gt C & ltu & gt & ltu & gt & ltu & gt. Respectively converting pET-ELP-N and pET-NpuC-Lunasin into Escherichia coli, optimizing conditions of recombinant protein expression, mixing, reverse phase transition circulating precipitation and Npu self-cracking, and finally obtaining unlabeled Lunasin (native recombination Lunasin). The purification method mediated by the ELP label has the advantages of low cost, relatively simple operation, economy and saving, and the purified recombinant polypeptide does not carry any label protein, thereby avoiding the immune phenomenon of an organism to the target protein in clinical application. The establishment of the expression and purification system of the bioactive peptide NpuC-Lunasinn provides an experimental basis for the future large-scale low-cost production of the bioactive peptide by Lunasin.
Drawings
FIG. 1 is lunasin PCR agarose electrophoresis;
lane 1: DL2000 marker; lanes 2, 3, 4, 5: PCR results for lunasin gene (175 bp);
FIG. 2 is a PCR identification of pMD19-T-Lunasin recombinant plasmid T vector (2% Agarose);
lane 1: DL2000 marker; lanes 2, 3, 4, 5, 6: PCR results of the pMD19-T-Lunasin recombinant plasmid T vector;
FIG. 3A is a diagram showing the results of amplification of Lunasin and Npuc genes, M: marker; lane 1: NpuC; lane 2: lunasin;
b is a PCR result chart of the recombinant NpuC-Lunasin fusion gene, and M: marker; lanes 1, 2, 3, 4: the PCR result of the recombinant NpuC-Lunasin (263bp) fusion gene;
FIG. 4A PCR identification result of the ligation of NpuC-Lunasin gene T vector, M: marker; lane 1: the result of the PCR product of NpuC-Lunasin (263 bp);
b is the double enzyme digestion identification result of the connection condition of the NpuC-Lunasin gene and the T vector, and M: marker; lanes 1, 2: double enzyme digestion results;
FIG. 5A shows the results of colony PCR, M: marker; lane 1: NpuC-Lunasin;
b is the result of double enzyme digestion identification of the expression vector, M: marker; lane 1: the enzyme digestion result of the NpuC-Lunasin recombinant expression vector;
FIG. 6 is a diagram showing the results of expression and purification of recombinant protein ELP-N;
m: protein marker; lanes 1-3: before IPTG induction; lanes 4-8: after inducing for 6h by 0.5mM IPTG; lane 9: the results of Westen-blot analysis after 6h induction by 0.5mM IPTG;
FIG. 7 shows the expression and purification results of NpuC-Lunasin;
m: protein marker; lanes 1-3: before IPTG induction; lanes 4-8: after 6h of induction with 0.5mM IPTG;
FIG. 8 shows the results of lunasin purification;
m: protein marker; lane 1: pre-purifying ELP-N; lane 2: soluble lysate of NpuC-Lunasin; lane 3: samples from lanes 1 and 2; lane 4: supernatant after precipitation of the ELP complex; lane 5: a mixture of ELP-N and NpuC-Lunasin for 0 h; lane 6: mixing the mixture of ELP-N and NpuC-Lunasin at 22 deg.C for 3 h; lane 7: ELP precipitation after 3 hours; lanes 8-9: after 3h the supernatant containing Lunasin was precipitated with ammonium sulphate.
Detailed Description
The present invention will be further described with reference to the following detailed description.
A non-column low-cost preparation method of food-derived soybean bioactive peptide Lunasin comprises the following steps:
1. food-derived soybean bioactive peptide lunasin gene obtaining method
(1) Obtaining target gene by PCR reaction
The Lunasin-encoding Gene was codon-optimized and synthesized using OPTIMIZER and Gene Designer according to Lunasin amino acid sequence published on PDB, and delivered to shanghai bio corporation for synthesis. The upstream and downstream primers are used for carrying out PCR amplification on the lunasin gene, the PCR system is 50ul, and the reaction conditions are as follows: 5min at 94 ℃; (94 ℃ for 30 s; 50 ℃ for 30 s; 72 ℃ for 1.5min)30 cycles; 10min at 72 ℃. And the PCR product is identified by 2% agarose gel electrophoresis and then recovered by a gel recovery kit to obtain the lunasin gene. As a result, as shown in FIG. 1, the lunasin gene amplified by PCR was analyzed by agarose gel electrophoresis, and the band size was 175bp, which was consistent with the size issued by GeneBank.
(2) TA cloning of Lunsian Gene fragments
The lunasin gene fragment was ligated into pMD19-T simple vector as: pMD19-T Simple Vector, 1. mu.l (50 ng); gene of interest, 2ul (30 ng); H2O, 1 ul; solution, 5. mu.l. Then, the cells were transformed into DH5 competent bacteria, plated with resistant LB plates, and the colonies obtained were identified by PCR, as shown in FIG. 2. And selecting positive clones, carrying out amplification culture, and then sending the positive clones to Shanghai bioengineering Co. The sequencing result is compared with the Lunasin sequence provided by GenBank, and the recombinant plasmid is named as PMD19-T-Lunasin after the sequence is identified to be correct.
2. Column-free lunasin purification
(1) Construction and identification of NpuC-Lunasin fusion gene cloning vector
1) lunasin amplification
The Lunasin is amplified by using upstream and downstream primers by taking PMD19-T-Lunasin containing Lunasin as a template. The PCR system is 50ul, and the reaction conditions are as follows: 5min at 94 ℃; (94 ℃ for 30 s; 50 ℃ for 30 s; 72 ℃ for 1.5min)30 cycles; 10min at 72 ℃. The PCR product was identified by 2% agarose gel electrophoresis, and the results are shown in FIG. 3A.
2) NpuC amplification
NpuC was amplified using upstream and downstream primers using NpuC-containing pET-NpuC-GFP as a template. The PCR system is 50ul, and the reaction conditions are as follows: 5min at 94 ℃; (94 ℃ for 30 s; 50 ℃ for 30 s; 72 ℃ for 1.5min)30 cycles; 10min at 72 ℃. The PCR product was identified by 2% agarose gel electrophoresis, and the results are shown in FIG. 3B.
3) NpuC-Lunasin fusion gene amplification
Using an upstream primer of the NpuC and a downstream primer of Lunasin to perform overlap on the NpuC and the Lunasin, and using the result as a template to perform PCR amplification, wherein the PCR system is 50ul, and the reaction conditions are as follows: 5min at 94 ℃; (94 ℃ for 30 s; 55 ℃ for 30 s; 72 ℃ for 1min)30 cycles; 10min at 72 ℃. The PCR product (NpuC-Lunasin) was identified by 2% agarose gel electrophoresis, and the results are shown in FIG. 4A.
4) TA cloning of fusion gene NpuC-Lunasin
The NpuC-Lunasin gene was ligated to T1-simple, the linker was 2ul of the desired gene, 1. mu. l T1-simple, and reacted at 25 ℃ for 5 min. Then, the cells were transformed into DH5 competent bacteria, plated with resistant LB plates, and the colonies obtained were identified by PCR, as shown in FIG. 4B. And selecting positive clones, carrying out amplification culture, and then sending the positive clones to Shanghai bioengineering Co. The Sequence results were analyzed using Sequence software and aligned to known sequences. After the sequence is identified to be correct, the recombinant plasmid is named as PMD 19-T-NpuC-Lunasin.
(2) Construction and identification of fusion gene NpuC-Lunasin expression vector
Coli DH5 containing PMD19-T-NpuC-Lunasin and pET26-b (+) plasmids was separately grown up and extracted, followed by double digestion with Nde I and Xho I. And cutting the gel, recovering the fragments of the enzyme digestion products NpuC-Lunasin and pET26-b (+) target fragments, and connecting at 16 ℃ overnight according to the molar ratio of 1: 10. The ligation products were transformed into DH5 competent cells (100ul) and plated on ampicillin-resistant LB solid medium at a concentration of 50 g/mL. After culturing for 16h, selecting positive colonies for amplification culture, carrying out colony PCR (shown in figure 5A) and Nde I and Xho I double-enzyme digestion identification (shown in figure 5B), and confirming that the size of the band is consistent with that of the recombinant protein gene, thereby proving that the connection is successful. The successfully constructed recombinant expression plasmid was named pET 26-NpuC-Lunasin.
(3) NpuC-Lunasin polypeptide expression
The positive strain containing pET26-NpuC-Lunasin plasmid is inoculated into LB culture medium, cultured at 37 ℃ and induced to express for 6h by 0.5mM IPTG. The cells were centrifuged at 6000rpm, the supernatant was discarded, and the cells were analyzed by TiCine/SDS-PAGE as shown in FIG. 6. Collecting wet bacteria according to the proportion that 10mL of 20mM Tris-HCL 7.4buffer (containing PMSF) is used for each gram of wet bacteria, carrying out ultrasonic disruption, and centrifuging to collect supernatant.
(4) purification of pET26-NpuC-Lunasin polypeptide
1) Expression and purification of ELP-N
The strain containing pET-ELP-N is inoculated into LB culture medium and cultured at 37 ℃, and the expression is induced by 0.5mM IPTG for 6 h. The bacterial solution was centrifuged at 6000rpm, the supernatant was discarded, and the cells were analyzed by TiCine/SDS-PAGE and Western-blot as shown in FIG. 7. Collecting wet bacteria, adding 10mL of 20mM Tris-HCl 7.4buffer (containing PMSF), carrying out ultrasonication, and centrifuging to collect supernatant. Centrifuging the lysate of the recombinant bacteria to obtain supernatant and the same volume of 0.8mol/L (NH)4)2S04Mixing, incubating at 30 ℃ for 10min, centrifuging at 14000g for 5min, and collecting the centrifugal precipitate to obtain the primary purified ELP-N fusion protein.
2) ELP-mediated purification of NpuC-Lunasin
Mixing the pre-purified ELP-N with the NpuC-Lunasin ultrasonic supernatant, incubating for 10min at room temperature, and mixing the supernatant with equal volume of 0.8mol/L (NH)4)2S04Mixing, incubating at 30 deg.C for 10min, centrifuging at 14000g for 5min, and collecting the precipitate. Adding 50mM DTT cutting fluid into the precipitate, incubating at room temperature for 3h, and adding equal volume of 0.8mol/L (NH)4)2S04After incubation for 10min, 14000g was centrifuged for 5min, and samples of the supernatant and pellet were taken and analyzed by TiCine/SDS-PAGE as shown in FIG. 8.
Sequence listing
<110> university of Dalian
<120> column-free low-cost preparation method of food-derived soybean bioactive peptide Lunasin
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tctaaatggc agcaccagca ggactcttgc cgtaaacagc tgcagggtgt taacctgacc 60
ccgtgcgaaa aacac 75
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ttactagtca tcgtcatcgt catcgtcatc gtcaccacga ccctggattt tttccatgat 60
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tagcttctaa ttctaaatgg cagcaccagc ag 32
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ccgctcgagt tactagtcat cgtcatcgtc atcg 34
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ttagaaggca tatgatcaaa atagccacac gtaaatattt agg 43
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tctaaatggc agcaccagca ggactcttgc cgtaaacagc tgcagggtgt taacctgacc 60
ccgtgcgaaa aacacatcat ggaaaaaatc cagggtcgtg gtgacgatga cgatgacgat 120
gacgatgac 129
Claims (1)
1. A low-cost and column-free preparation method of food-derived soybean bioactive peptide lunasin is characterized by comprising the following steps: acquiring a Lunasin gene fragment by adopting an Overlapping Extension PCR method, and realizing the fusion expression of the Lunasin gene fragment and the C end of DnaE split intein from Nostoc punctiform; fusing and expressing an ELP label of the elastin-like protein and the N end of a DnaE split intein; finally, obtaining the natural N-terminal recombinant Lunasin polypeptide by utilizing trans-splicing of the broken intein and reversible cycle phase change of the ELP;
the preparation method comprises the following steps:
(1) obtaining of food-derived soybean bioactive peptide lunasin gene
According to the Lunasin sequence, synthesizing two-end primers, an upstream primer:
5-TCTAAATGGCAGCACCAGCAGGACTCTTGCCGTAAACAGCTGCAGGGTGTTAACCTGACCCCGTGCGAAAAACACATC-3, downstream primer: 5-TTACTAGTCATCGTCATCGTCATCGTCATCGTCACCACGACCCTGGATTTTTTCCATGATGTGTTTTTCGCACGGGGTC-3, performing overlapping extension PCR on the upstream and downstream primers to obtain Lunasin, connecting the Lunasin gene to pMD19-T simple vector, then transforming to DH5 competent bacteria, coating an LB plate containing resistance, performing PCR identification on the obtained colony, selecting positive clone, performing amplification culture sequencing, and naming the recombinant plasmid as pMD19-T-Lunasin after the identification sequence is correct;
(2) construction of NpuC-Lunasin fusion gene expression vector
Based on Lunasin and DnaE sequences, primers are designed, wherein an upstream primer 1: TAGCTTCTAATTCTAAATGGCAGCACCAGCAG, downstream primer 1: CCGCTCGAGTTACTAGTCATCGTCATCGTCATCG, upstream primer 2: TTAGAAGGCATATGATCAAAATAGCCACACGTAAATATTTAGG, downstream primer 2: GCTGGTGCTGCCATTTAGAATTAGAAGCTATGAAGCCATTTTTG, using a T vector pMD19-T-Lunasin containing the Lunasin fragment as a template, and carrying out PCR amplification on the lunsian gene by using an upstream primer 1 and a downstream primer 1; taking pET-NpuC-GFP plasmid as a template, and carrying out PCR amplification on the NpuC gene by using an upstream primer 2 and a downstream primer 2; respectively taking the NpuC and the Lunasin templates, using the upstream primer 2 and the downstream primer 1 to perform overlap, and taking the result as the template to perform PCR amplification to obtain the NpuC-Lunasin gene; the gene of the NpuC-Lunasin is connected to a pMD19-T simple vector, then is transformed into DH5 competent bacteria, is coated with an LB plate containing resistance, the obtained colony is subjected to PCR identification, and the recombinant plasmid is named as PMD19-T-NpuC-Lunasin after the amplified culture sequencing identification sequence of the selected positive clone is correct;
(3) inducible expression of pET-26-NpuC-Lunasin polypeptide
Inoculating a positive strain containing pET26-NpuC-Lunasin plasmid into an LB culture medium, culturing at 37 ℃, carrying out induced expression for 6h by 0.5mMIPTG, and collecting supernatant, namely NpuC-Lunasin;
(4) expression and purification of ELP-N to obtain pre-purified ELP-N fusion protein;
inoculating a bacterial strain containing pET-ELP-N into an LB culture medium for culture at 37 ℃, inducing expression for 6h by 0.5mM IPTG, collecting supernatant, mixing with equal volume of 0.8mol/L (NH4)2S04, incubating for 10min at 30 ℃, centrifuging for 5min at 14000g, collecting centrifugal precipitate to obtain preliminarily purified ELP-N fusion protein;
(5) ELP-mediated purification of NpuC-Lunasin
Mixing the pre-purified ELP-N with the NpuC-Lunasin ultrasonic supernatant, and mixing the supernatant mixed solution with 0.8mol/L (NH4)2S04 with the same volume; incubating at 30 deg.C for 10min, and centrifuging at 14000g for 5 min; collecting the centrifugal precipitate in optimized cutting fluid 50mM DTT, incubating for 3h at room temperature, adding equal volume of 0.8mol/L (NH4)2S04, incubating for 10min, centrifuging for 5min at 14000g, and taking the supernatant as the Lunasin protein containing the natural N-terminal.
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| EP1173476A1 (en) * | 1999-04-30 | 2002-01-23 | The Regents Of The University Of California | Soybean protein nutraceuticals |
| CN101418290A (en) * | 2008-12-02 | 2009-04-29 | 中山大学 | High efficiency ELP fusion protease as well as preparation and application thereof |
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| CN105647960A (en) * | 2016-03-28 | 2016-06-08 | 中国农业科学院作物科学研究所 | Inducible expression, purification and activity identification method of restructured lunasin polypeptide in pichia pastoris |
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| EP1173476A1 (en) * | 1999-04-30 | 2002-01-23 | The Regents Of The University Of California | Soybean protein nutraceuticals |
| CN101418290A (en) * | 2008-12-02 | 2009-04-29 | 中山大学 | High efficiency ELP fusion protease as well as preparation and application thereof |
| CN103993004A (en) * | 2014-01-21 | 2014-08-20 | 舟山出入境检验检疫局综合技术服务中心 | Cloning and expression method for Ani s4 antigen gene of Anisakis simplex |
| CN105647960A (en) * | 2016-03-28 | 2016-06-08 | 中国农业科学院作物科学研究所 | Inducible expression, purification and activity identification method of restructured lunasin polypeptide in pichia pastoris |
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Application publication date: 20180515 Assignee: Dalian Zhicheng Biotechnology Co.,Ltd. Assignor: DALIAN University Contract record no.: X2023210000188 Denomination of invention: A columnless and low-cost preparation method for the bioactive peptide Lunasin from foodborne soybeans Granted publication date: 20210330 License type: Common License Record date: 20231123 |
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Application publication date: 20180515 Assignee: Dalian darente fragrance Technology Co.,Ltd. Assignor: DALIAN University Contract record no.: X2023210000245 Denomination of invention: A columnless and low-cost preparation method for the bioactive peptide Lunasin from foodborne soybeans Granted publication date: 20210330 License type: Common License Record date: 20231129 Application publication date: 20180515 Assignee: Dalian Fengrui Food Processing Co.,Ltd. Assignor: DALIAN University Contract record no.: X2023210000226 Denomination of invention: A columnless and low-cost preparation method for the bioactive peptide Lunasin from foodborne soybeans Granted publication date: 20210330 License type: Common License Record date: 20231129 |
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