CN115093470B - Intein Mtu RecA mutant and application thereof in production of glutathione GSH - Google Patents
Intein Mtu RecA mutant and application thereof in production of glutathione GSH Download PDFInfo
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Abstract
The invention relates to an intein Mtu RecA mutant, which is a RecA protein derived from Mycobacterium tuberculosis, the sequence of the RecA protein is shown as SEQ ID No.3, the coding DNA is shown as SEQ ID No.4, and pET28 a-intein Mtu RecA mutant-glutathione GSH and a prokaryotic expression vector thereof are constructed by utilizing the intein. Also disclosed is a method for producing glutathione GSH using the intein. The invention discloses an intein Mtu RecA mutant, and fusion expression is carried out by the intein and glutathione GSH, the expression mode has the advantages of high expression quantity and simple purification, and can greatly reduce the production cost of the glutathione GSH. Compared with the traditional intein Mtu RecA, the intein Mtu RecA mutant can obviously improve the yield and the purity of glutathione GSH.
Description
Technical Field
The invention belongs to the technical field of protein engineering, and particularly relates to an intein Mtu RecA mutant which can be applied to production of glutathione GSH.
Background
Glutathione (GSH) is a tripeptide containing gamma-amide bonds and sulfhydryl groups, and is composed of glutamic acid, cysteine and glycine. The sulfydryl on the cysteine is an active group of the cysteine, and is easy to combine with certain medicines, toxins and the like, so that the cysteine has an integrated detoxification function. The glutathione can be used for medicines, can be used as a base material of functional foods, is widely applied to the functional foods such as anti-aging, immunity enhancement, anti-tumor and the like, can eliminate free radicals in vivo, has an anti-oxidation function, and can help to keep the functions of a normal immune system. Therefore, the glutathione GSH has important market application value. However, the current industrial synthesis of glutathione mainly depends on chemical synthesis, and the synthesis method has the disadvantages of many byproducts, low yield, high pollution and high cost, and is difficult to meet the requirement of carbon neutralization. The biosynthesis can effectively solve the defects of chemical synthesis, and has the advantages of high yield, low cost, energy conservation, environmental protection and the like. However, there is still no efficient synthetic route for glutathione GSH of complex structure.
Disclosure of Invention
The invention aims to provide an intein Mtu RecA mutant, and the mutant is applied to expression, separation and purification of glutathione GSH.
In order to achieve the above object, the technical solutions adopted are as follows:
an intein Mtu RecA mutant is derived from RecA protein of Mycobacterium tuberculosis (Sequence ID: CFE 66777.1), two amino acid sequences of 93-186 and 488-531 are intercepted to form a fusion protein, the first cysteine is replaced by alanine, an asparagine is added at the C terminal, the specific amino acid Sequence of the mutant intein Mtu RecA is shown in SEQ ID No.3, and the coding DNA Sequence can be a Sequence shown in SEQ ID No.4 in a Sequence table or a polynucleotide Sequence of the protein Sequence shown in SEQ ID No.3 in the Sequence table.
The invention also discloses application of the intein Mtu RecA mutant in production of glutathione GSH.
The invention also discloses an intein Mtu RecA mutant-glutathione GSH fusion protein, the sequence of which is shown in SEQ ID No. 7.
The sequence of the coding DNA of the intein Mtu RecA mutant-glutathione GSH fusion protein can be a sequence shown by SEQ ID No.8 in a sequence table, or a polynucleotide sequence of a protein sequence shown by SEQ ID No.7 in the sequence table.
The invention also discloses a pET28 a-intein Mtu RecA mutant-glutathione GSH expression plasmid for expressing the intein Mtu RecA mutant-glutathione GSH fusion protein.
The prokaryotic expression vector of the intein Mtu RecA mutant-glutathione GSH fusion protein.
Preferably, the prokaryotic expression vector is formed by transforming the pET28 a-intein Mtu RecA mutant-glutathione GSH expression plasmid into an escherichia coli host bacterium.
The invention also discloses a production method of the glutathione GSH, which is characterized by comprising the following steps:
(1) Constructing pET28 a-intein Mtu RecA mutant-glutathione GSH expression plasmid;
(2) Converting the pET28 a-intein Mtu RecA mutant-glutathione GSH expression plasmid into escherichia coli host bacteria, and selecting positive clone;
(3) Culturing positive clone, inducing expression of glutathione GSH;
(4) Separating and purifying glutathione GSH. The invention has the advantages over the prior art that:
the invention discloses an intein Mtu RecA mutant, and fusion expression is carried out by the intein and glutathione GSH, the expression mode has the advantages of high expression quantity and simple purification, and the production cost of the glutathione GSH can be greatly reduced. Compared with the traditional intein Mtu RecA, the intein Mtu RecA mutant can obviously improve the yield and the purity of glutathione GSH.
Drawings
FIG. 1: the results of sequence alignment of the classical intein Mtu RecA with the mutant intein Mtu RecA are shown without asparagine at the C-terminus of the mutant intein Mtu RecA.
FIG. 2: the fusion protein of traditional intein Mtu RecA and intein Mtu RecA mutant and GSH induces expression and purification result.
FIG. 3: HPLC detects the effect of releasing GSH by the cleavage of the traditional intein Mtu RecA.
FIG. 4 is a schematic view of: HPLC detects the effect of release of GSH by cleavage of the intein Mtu RecA mutant.
Detailed Description
The invention is further illustrated below with reference to specific examples, which should not be construed as in any way limiting the scope of the application of the invention. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 design of intein Mtu RecA mutants
The 93-531 rd amino acid Sequence was truncated from the RecA protein of Mycobacterium tuberculosis (Sequence ID: CFE 66777.1) based on alignment and search of intein sequences. The amino acid sequence between 187-487 was then removed based on the alignment of the amino acid sequences of the mini-inteins, and the final alignment with the conventional intein Mtu RecA is shown in FIG. 1. The amino acid sequence and DNA sequence of the traditional intein Mtu RecA are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. Furthermore, we replaced the first cysteine at the N-terminus of the truncated Mtu RecA intein with alanine, and removed the N-terminal self-cleavage activity of the intein variant; the addition of an asparagine to the C-terminus of the intein activates the self-cleaving activity of the C-terminus of the intein. The amino acid sequence of the final Mtu RecA intein variant is shown in SEQ ID NO.3 and the DNA sequence is shown in SEQ ID NO.4. Meanwhile, the protein tertiary structures of the traditional Mtu RecA intein and the intein variant are predicted by using AlphaFold2, and sequence comparison is carried out, so that the protein tertiary structures of the intein before and after mutation are highly similar, the active center of the intein is not influenced, and the original intein structure is still maintained.
Example 2 construction of intein Mtu RecA mutant and glutathione GSH fusion protein prokaryotic expression vector
The amino acid sequence of the traditional intein Mtu RecA and glutathione GSH fusion protein is shown in SEQ ID NO.5, and the DNA sequence is shown in SEQ ID NO.6. The amino acid sequence of the intein Mtu RecA mutant and glutathione GSH fusion protein is shown in SEQ ID NO.7, and the DNA sequence is shown in SEQ ID NO.8. The gene sequence is synthesized in Nanjing Kingsrei Biotechnology, limited liability company, the 5 'end enzyme cutting site is BamH I, and the 3' end enzyme cutting site is Xho I. After 1ug of the synthesized plasmid and pET28a were cleaved by BamH I and XhoI double digestion, fragments of the corresponding size were recovered on a 2% agarose gel. The fusion sequence of the classical intein Mtu RecA or mutant intein Mtu RecA and glutathione GSH was then ligated to the cut linearized pET28a backbone using T4 DNA ligase. The ligated product was transformed into BL21 (DE 3) E.coli competence, spread on solid LB plates containing kanamycin, and cultured overnight at 37 ℃. Selecting a monoclonal antibody to be in an LB liquid culture medium containing kanamycin, and performing PCR identification on the bacterial liquid by using a universal primer of a carrier T7 promoter after shake culture. And (4) performing first-generation sequencing on the successfully identified bacterial liquid by using a universal primer, and storing the correctly sequenced bacterial strain.
Example 3 inducible expression of the intein Mtu RecA and variants and glutathione GSH fusion proteins
The constructed plasmid is transformed into a BL21 (DE 3) expression strain, a monoclonal strain is picked up and put into an LB culture medium, the OD600 value in the amplification culture is 0.8, 1/20 volume of 1M Tris-HCl buffer solution (pH 8.5) and IPTG with the final concentration of 1mM are added, and the culture is continued for 4-6h at 37 ℃ and 200 rpm. The cells were centrifuged at 10000rpm and 4 ℃ for 20min to collect the cells. SDS-PAGE detects the result of the induced expression of the fusion protein. The result is shown in figure 2, IPTG can efficiently induce the expression of intein Mtu RecA and mutant and glutathione GSH fusion protein, and accounts for about 40 percent of the total protein content.
Example 4 purification of glutathione GSH
This example discloses specific steps of a method for purification of glutathione GSH.
1) Inducing expression: the constructed plasmid is transformed into a BL21 (DE 3) expression strain, a monoclonal strain is picked up and put into an LB culture medium, the OD600 value in the amplification culture is 0.8, 1/20 volume of 1M Tris-HCl buffer solution (pH 8.5) and IPTG with the final concentration of 1mM are added, and the culture is continued for 4-6h at 37 ℃ and 200 rpm. The cells were centrifuged at 10000rpm and 4 ℃ for 20min to collect the cells. The mycelia were washed 2 times with PBS.
2) And (3) crushing thalli: the cells were resuspended in lysis buffer (20mM Tris,500mM NaCl, pH 8.0), disrupted using pressure or sonication, and stained by microscopy until no cells were evident. 12000rpm,4 degrees centrifugal 20min, collect the supernatant, 0.45um filter membrane filtration.
3) Affinity chromatography: after the Ni-NTA affinity chromatography column was fully equilibrated with lysis buffer, the filtered bacterial lysate was added at a flow rate of 0.5ml/min, followed by full washing with lysis buffer containing 20mM imidazole.
4) Intein cleavage: 50mM phosphate buffer pH 6.0 was added thereto, and after mixing, the mixture was left overnight at room temperature to collect a flow-through solution. The intein was then eluted with 200mM imidazole lysis buffer.
5) And detecting the purification result by HPLC.
The results are shown in FIGS. 3-4, and the intein Mtu RecA and mutant and glutathione GSH fusion protein can be efficiently enriched by using the affinity tag. After the intein is cut on the medium, the content of glutathione GSH released into the circulating liquid by the traditional Mtu RecA intein cutting can reach 2.1mg yield per liter of bacterial liquid, and the purity is more than 85%. The content of glutathione GSH released into a circulating liquid by the cutting of the intein Mtu RecA mutant can reach 11mg yield per liter of bacterial liquid, and the purity is more than 98 percent. The results show that the screened intein Mtu RecA mutant can quickly and efficiently express and purify glutathione GSH.
Sequence listing
<110> Dry-phase Biotechnology Ltd of Guangzhou City
<120> intein Mtu RecA mutant and application thereof in production of glutathione GSH
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 167
<212> PRT
<213> Mycobacterium tuberculosis
<400> 1
Cys Leu Ala Glu Gly Thr Arg Ile Phe Asp Pro Val Thr Gly Thr Thr
1 5 10 15
His Arg Ile Glu Asp Val Val Asp Gly Arg Lys Pro Ile His Val Val
20 25 30
Ala Ala Ala Lys Asp Gly Thr Leu His Ala Arg Pro Val Val Ser Trp
35 40 45
Phe Asp Gln Gly Thr Arg Asp Val Ile Gly Leu Arg Ile Ala Gly Gly
50 55 60
Ala Ile Leu Trp Ala Thr Pro Asp His Lys Val Leu Thr Glu Tyr Gly
65 70 75 80
Trp Arg Ala Ala Gly Glu Leu Arg Lys Gly Asp Arg Val Ala Gln Pro
85 90 95
Arg Arg Phe Asp Gly Phe Gly Asp Ser Ala Pro Ile Pro Ala Arg Val
100 105 110
Gln Ala Leu Ala Asp Ala Leu Asp Asp Lys Phe Leu His Asp Met Leu
115 120 125
Ala Glu Glu Leu Arg Tyr Ser Val Ile Arg Glu Val Leu Pro Thr Arg
130 135 140
Arg Ala Arg Thr Phe Asp Leu Glu Val Glu Glu Leu His Thr Leu Val
145 150 155 160
Ala Glu Gly Val Val Val His
165
<210> 2
<211> 501
<212> DNA
<213> Mycobacterium tuberculosis
<400> 2
tgtctggcgg aaggtacccg tatttttgac ccggttactg gtaccacgca ccgcatcgaa 60
gatgtcgtag acggtcgtaa accgattcac gtagttgctg cggctaaaga cggcaccctg 120
catgcgcgtc cggttgtttc ttggttcgac cagggtactc gtgatgtcat cggcctgcgt 180
atcgcgggtg gtgcaatcct gtgggctact ccggatcata aagtactgac tgagtacggt 240
tggcgtgccg ctggtgagct gcgtaaaggt gaccgtgtag cgcagccgcg tcgttttgac 300
ggtttcggcg attccgctcc gatcccagct cgtgtacagg ctctggccga tgcactggat 360
gacaaattcc tgcacgatat gctggcggaa gaactgcgct acagcgtaat ccgcgaagtt 420
ctgccaaccc gccgtgctcg taccttcgac ctggaagtcg aggaactgca cactctggtt 480
gctgaaggtg tggttgttca c 501
<210> 3
<211> 139
<212> PRT
<213> Artifical Sequence
<400> 3
Ala Leu Ala Glu Gly Thr Arg Ile Phe Asp Pro Val Thr Gly Thr Thr
1 5 10 15
His Arg Ile Glu Asp Val Val Asp Gly Arg Lys Pro Ile His Val Val
20 25 30
Ala Ala Ala Lys Asp Gly Thr Leu His Ala Arg Pro Val Val Ser Trp
35 40 45
Phe Asp Gln Gly Thr Arg Asp Val Ile Gly Leu Arg Ile Ala Gly Gly
50 55 60
Ala Ile Val Trp Ala Thr Pro Asp His Lys Val Leu Thr Glu Tyr Gly
65 70 75 80
Trp Arg Ala Ala Gly Glu Leu Arg Lys Gly Asp Arg Val Ala Leu His
85 90 95
Asp Met Leu Ala Glu Glu Leu Arg Tyr Ser Val Ile Arg Glu Val Leu
100 105 110
Pro Thr Arg Arg Ala Arg Thr Phe Asp Leu Glu Val Glu Glu Leu His
115 120 125
Thr Leu Val Ala Glu Gly Val Val Val His Asn
130 135
<210> 4
<211> 417
<212> DNA
<213> Artifical Sequence
<400> 4
gcgctggctg agggtactcg tatttttgat ccggttactg gtaccaccca tcgtatcgag 60
gatgttgttg acggccgtaa accgatccac gtagtagcgg ctgccaaaga cggtactctg 120
cacgcgcgtc cggttgtttc ttggttcgac cagggtactc gtgatgttat cggcctgcgc 180
attgcaggtg gtgctatcgt ttgggcaacc ccggaccaca aagtactgac cgaatatggt 240
tggcgtgctg ccggtgaact gcgcaagggt gatcgtgttg ctctgcatga tatgctggct 300
gaggaactgc gctactccgt cattcgcgaa gttctgccaa cccgtcgtgc acgtaccttc 360
gatctggaag tagaagaact gcacaccctg gttgctgaag gcgtggttgt tcacaat 417
<210> 5
<211> 170
<212> PRT
<213> Artifical Sequence
<400> 5
Ala Leu Ala Glu Gly Thr Arg Ile Phe Asp Pro Val Thr Gly Thr Thr
1 5 10 15
His Arg Ile Glu Asp Val Val Asp Gly Arg Lys Pro Ile His Val Val
20 25 30
Ala Ala Ala Lys Asp Gly Thr Leu His Ala Arg Pro Val Val Ser Trp
35 40 45
Phe Asp Gln Gly Thr Arg Asp Val Ile Gly Leu Arg Ile Ala Gly Gly
50 55 60
Ala Ile Leu Trp Ala Thr Pro Asp His Lys Val Leu Thr Glu Tyr Gly
65 70 75 80
Trp Arg Ala Ala Gly Glu Leu Arg Lys Gly Asp Arg Val Ala Gln Pro
85 90 95
Arg Arg Phe Asp Gly Phe Gly Asp Ser Ala Pro Ile Pro Ala Arg Val
100 105 110
Gln Ala Leu Ala Asp Ala Leu Asp Asp Lys Phe Leu His Asp Met Leu
115 120 125
Ala Glu Glu Leu Arg Tyr Ser Val Ile Arg Glu Val Leu Pro Thr Arg
130 135 140
Arg Ala Arg Thr Phe Asp Leu Glu Val Glu Glu Leu His Thr Leu Val
145 150 155 160
Ala Glu Gly Val Val Val His Gly Ser His
165 170
<210> 6
<211> 510
<212> DNA
<213> Artifical Sequence
<400> 6
tgtctggcgg aaggtacccg tatttttgac ccggttactg gtaccacgca ccgcatcgaa 60
gatgtcgtag acggtcgtaa accgattcac gtagttgctg cggctaaaga cggcaccctg 120
catgcgcgtc cggttgtttc ttggttcgac cagggtactc gtgatgtcat cggcctgcgt 180
atcgcgggtg gtgcaatcct gtgggctact ccggatcata aagtactgac tgagtacggt 240
tggcgtgccg ctggtgagct gcgtaaaggt gaccgtgtag cgcagccgcg tcgttttgac 300
ggtttcggcg attccgctcc gatcccagct cgtgtacagg ctctggccga tgcactggat 360
gacaaattcc tgcacgatat gctggcggaa gaactgcgct acagcgtaat ccgcgaagtt 420
ctgccaaccc gccgtgctcg taccttcgac ctggaagtcg aggaactgca cactctggtt 480
gctgaaggtg tggttgttca cggtagccat 510
<210> 7
<211> 142
<212> PRT
<213> Artifical Sequence
<400> 7
Ala Leu Ala Glu Gly Thr Arg Ile Phe Asp Pro Val Thr Gly Thr Thr
1 5 10 15
His Arg Ile Glu Asp Val Val Asp Gly Arg Lys Pro Ile His Val Val
20 25 30
Ala Ala Ala Lys Asp Gly Thr Leu His Ala Arg Pro Val Val Ser Trp
35 40 45
Phe Asp Gln Gly Thr Arg Asp Val Ile Gly Leu Arg Ile Ala Gly Gly
50 55 60
Ala Ile Val Trp Ala Thr Pro Asp His Lys Val Leu Thr Glu Tyr Gly
65 70 75 80
Trp Arg Ala Ala Gly Glu Leu Arg Lys Gly Asp Arg Val Ala Leu His
85 90 95
Asp Met Leu Ala Glu Glu Leu Arg Tyr Ser Val Ile Arg Glu Val Leu
100 105 110
Pro Thr Arg Arg Ala Arg Thr Phe Asp Leu Glu Val Glu Glu Leu His
115 120 125
Thr Leu Val Ala Glu Gly Val Val Val His Asn Gly Ser His
130 135 140
<210> 8
<211> 426
<212> DNA
<213> Artifical Sequence
<400> 8
gcgctggctg agggtactcg tatttttgat ccggttactg gtaccaccca tcgtatcgag 60
gatgttgttg acggccgtaa accgatccac gtagtagcgg ctgccaaaga cggtactctg 120
cacgcgcgtc cggttgtttc ttggttcgac cagggtactc gtgatgttat cggcctgcgc 180
attgcaggtg gtgctatcgt ttgggcaacc ccggaccaca aagtactgac cgaatatggt 240
tggcgtgctg ccggtgaact gcgcaagggt gatcgtgttg ctctgcatga tatgctggct 300
gaggaactgc gctactccgt cattcgcgaa gttctgccaa cccgtcgtgc acgtaccttc 360
gatctggaag tagaagaact gcacaccctg gttgctgaag gcgtggttgt tcacaatggt 420
agccat 426
Claims (8)
1. An intein Mtu RecA mutant is characterized in that the sequence is shown as SEQ ID No. 3.
2. DNA encoding the mutant intein Mtu RecA according to claim 1, characterized in that:
a) A DNA sequence shown as SEQ ID No.4 in the sequence table;
b) A polynucleotide sequence of SEQ ID No.3 protein sequence in the coding sequence list.
3. An intein Mtu RecA mutant-glutathione GSH fusion protein is characterized in that the sequence is shown in SEQ ID No. 7.
4. The DNA encoding the intein Mtu RecA mutant-glutathione GSH fusion protein of claim 3, characterized in that:
a) A DNA sequence shown as SEQ ID No.8 in the sequence table;
b) A polynucleotide sequence of SEQ ID No.7 protein sequence in the coding sequence table.
5. A pET28 a-intein Mtu RecA mutant-glutathione GSH expression plasmid, characterized by expressing the intein Mtu RecA mutant-glutathione GSH fusion protein of claim 3.
6. The prokaryotic expression vector of the intein Mtu RecA mutant-glutathione GSH fusion protein of claim 3.
7. The prokaryotic expression vector according to claim 6, which is obtained by transforming the expression plasmid pET28 a-intein Mtu RecA mutant-glutathione GSH of claim 5 into E.coli host bacteria.
8. A method for producing glutathione GSH is characterized by comprising the following steps:
(1) Constructing pET28 a-intein Mtu RecA mutant-glutathione GSH expression plasmid;
(2) Converting the pET28 a-intein Mtu RecA mutant-glutathione GSH expression plasmid into escherichia coli host bacteria, and selecting positive clone;
(3) Culturing positive clone, and inducing the expression of intein Mtu RecA mutant-glutathione GSH fusion protein, wherein the sequence of intein Mtu RecA mutant-glutathione GSH fusion protein is shown as SEQ ID No. 7;
(4) Separating and purifying glutathione GSH.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104328092A (en) * | 2014-09-28 | 2015-02-04 | 邦泰生物工程(深圳)有限公司 | Glutathione synthetase mutant, encoding gene and application |
| CN115074340A (en) * | 2022-06-17 | 2022-09-20 | 广州市乾相生物科技有限公司 | Novel intein and application thereof in synthesis of human tropoelastin |
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| DK1141250T3 (en) * | 1998-12-18 | 2006-07-10 | Penn State Res Found | Intein mediates cyclization of peptides |
| WO2005098043A2 (en) * | 2004-03-30 | 2005-10-20 | The President And Fellows Of Harvard College | Ligand-dependent protein splicing |
| CN101886105A (en) * | 2009-05-12 | 2010-11-17 | 南京大学 | Preparation of human glucagon like peptide (GLP-1) by mini-intein mediation |
| CN101967493A (en) * | 2010-06-10 | 2011-02-09 | 广东医学院 | Prokaryotic expression vector and application thereof |
| GB201016742D0 (en) * | 2010-10-05 | 2010-11-17 | Health Prot Agency | Clostridium difficile antigens |
| US9394345B2 (en) * | 2013-03-15 | 2016-07-19 | Pioneer Hi-Bred International, Inc. | PHI-4 polypeptides and methods for their use |
| US10687542B2 (en) * | 2015-09-18 | 2020-06-23 | Agrivida, Inc. | Engineered phytases and methods of using the same |
| WO2020125707A1 (en) * | 2018-12-19 | 2020-06-25 | Tsinghua University | Mtu δi-cm intein variant and the use thereof |
| BR112021018606A2 (en) * | 2019-03-19 | 2021-11-23 | Harvard College | Methods and compositions for editing nucleotide sequences |
| CN113528479B (en) * | 2020-04-13 | 2023-01-24 | 中国科学院微生物研究所 | Efficient preparation method of transglutaminase and special engineering bacteria thereof |
| CN114075571A (en) * | 2020-08-18 | 2022-02-22 | 梦芊科技知识产权有限公司 | Nucleic acid constructs of epidermal growth factor, methods of production and compositions thereof |
| CN113549144A (en) * | 2021-06-30 | 2021-10-26 | 华南理工大学 | Method for producing and purifying teriparatide hPTH (1-34) |
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| CN104328092A (en) * | 2014-09-28 | 2015-02-04 | 邦泰生物工程(深圳)有限公司 | Glutathione synthetase mutant, encoding gene and application |
| CN115074340A (en) * | 2022-06-17 | 2022-09-20 | 广州市乾相生物科技有限公司 | Novel intein and application thereof in synthesis of human tropoelastin |
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