CN102492646A - Recombination escherichia coli for producing aminopeptidase with high yield and construction method of recombination escherichia coli - Google Patents
Recombination escherichia coli for producing aminopeptidase with high yield and construction method of recombination escherichia coli Download PDFInfo
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- CN102492646A CN102492646A CN2011103744043A CN201110374404A CN102492646A CN 102492646 A CN102492646 A CN 102492646A CN 2011103744043 A CN2011103744043 A CN 2011103744043A CN 201110374404 A CN201110374404 A CN 201110374404A CN 102492646 A CN102492646 A CN 102492646A
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Abstract
The invention discloses a recombination escherichia coli for producing aminopeptidase with high yield and a construction method of the recombination escherichia coli, belonging to the technical field of biological engineering. The construction method is simple and effective; and the constructed recombination Escherichia coli can effectively express aminopeptidase and provides required protein for researching characteristics and structures and the like of the aminopeptidase in the later stage.
Description
Technical field:
The present invention relates to a kind of recombination bacillus coli and construction process thereof of high yield aminopeptidase, belong to technical field of bioengineering.
Background technology:
Aminopeptidase be one type can protein hydrolysate and the circumscribed proteolytic ferment of polypeptide N terminal amino acid, extensively be present in plant-animal and the mikrobe.Aminopeptidase plays an important role in many biological processings, like proteinic digestion and decomposition, proteinic maturation, the adjusting of hormone secretion and the control of cell cycle.Undoubtedly, the initiation of numerous disease is all relevant with aminopeptidase, comprises inflammation, cataract, mellitus, apoplexy, cystic fibrosis even cancer and AIDS etc.Therefore, catalyst mechanism and some of correctly understanding aminopeptidase influence aminopeptidase and bring into normal play the factor of biological function to prevention with treat these diseases important effect is arranged.
At present, microbial fermentation is produced aminopeptidase and is had the problem that yields poorly, and the output that how to improve microbial method production aminopeptidase becomes the problem that presses for solution.
Summary of the invention:
Technical problem to be solved by this invention provides: a kind of recombination bacillus coli that efficiently expresses aminopeptidase, be with the removal that derives from subtilis Zj016 the aminopeptidase gene of signal peptide import the genetic engineering bacterium that E.coli BL21 (DE3) obtains.
Said subtilis Zj016 sees Tian Yaping for details, and the palpus beautiful jade is quick.A kind of purifying of subtilis aminopeptidase and zymologic property [J]. food and fermentation industries, 2006,32 (3): 7-10.
The said aminopeptidase nucleotide sequence of having removed signal peptide is shown in SEQ ID NO.1.
The technical problem that the present invention also will solve provides a kind of said construction process that efficiently expresses the bacillus coli gene engineering bacteria of aminopeptidase.
For solving the problems of the technologies described above, step is following:
1) collects the pure enzyme of subtilis Zj016 excretory and carry out N terminal amino acid sequence mensuration;
2) aminoacid sequence that records is compared in ncbi database; According to the nucleotide sequence of the protein-high of its consistence and design primer P1 and P2 clone aminopeptidase gene; With said aminopeptidase gene transformation E.coli JM109, obtain recombinant plasmid PUC19-SAP;
3) analyze the aminopeptidase nucleotide sequence, design primer P3 and P4 clone the gene of the encoding mature aminopeptidase that does not contain signal peptide; The gene of said ripe aminopeptidase is connected with carrier pET-28a (+), selects positive plasmid Transformed E .coli BL21 (DE3).
Said primer P1 nucleotide sequences is shown in SEQ ID NO.2.
Said primer P2 nucleotide sequences is shown in SEQ ID NO.3.
Said primer P3 nucleotide sequences is shown in SEQ ID NO.4.
Said primer P4 nucleotide sequences is shown in SEQ ID NO.5.
The invention provides a kind of recombination bacillus coli and construction process thereof of high yield aminopeptidase, this method is simply effective, and the recombination bacillus coli of structure can efficiently express aminopeptidase, and the characteristic, structure that can study aminopeptidase for the later stage etc. provides desirable proteins.
Description of drawings:
Fig. 1: the pure enzyme N end of wild bacterium sequencing result.
Fig. 2: the clone result (1: goal gene) of subtilis aminopeptidase gene.
Fig. 3: recombinant plasmid PUC19-SAP double digestion result, (1:PUC19,2: goal gene).
Fig. 4: recombinant plasmid pET-28a (+)-AP double digestion result (1:pET-28a (+); 2: goal gene).
Fig. 5: expression of recombinant e. coli SDS-PAGE result (M:marker; 1: do not induce; 2: induce; 3: target protein).
Embodiment:
Material and detection method
The LB substratum:: 1% Tryptones, 0.5% yeast extract, 1% sodium-chlor, pH 7.0.
TB substratum: 1.2% Tryptones, 2.4% yeast extract, 0.4% glycerine, 17mM KH
2PO
4, 72mMK
2HPO
4
The amino-peptidase activity measuring method: with L-leucine-p-Nitroaniline is substrate, in the Tris-HCl of 50mM pH 8.5 damping fluid, adds the enzyme liquid and the substrate (1mM) of dilution certain multiple, and 50 ℃ of water-bath 10min measure absorbancy under 405nm.Enzyme unit definition alive: under 50 ℃, it is enzyme unit (1U) alive that 1min decomposition L-leucine-p-Nitroaniline produces the required enzyme amount of 1 μ M p-Nitroaniline.
Embodiment 1:
After the pure enzyme of subtilis Zj016 excretory carried out the SDS-PAGE electrophoresis, take off gel, remove all concentrated glue from sheet glass.Gel is immersed electricity change 30min in the damping fluid (CAPS 10mM, methyl alcohol 10%, pH 11).Filter paper soaks 1min in electricity commentaries on classics damping fluid.Pvdf membrane is put in 5min in the distilled water with film after in methyl alcohol, soaking 15s, carefully is put in balance 10min in the transfering buffering liquid again.Open the transfer clip then, black orifice plate is put into clip with foam pad, filter paper, gel, film, filter paper, foam pad down in order.Transfer is folded up in the transfer groove, the negative pole of black orifice plate face back up pad, white orifice plate is aimed at the positive pole of back up pad.In transfer groove, add the whole submergences of appropriate amount of buffer solution to transfer groove.Electric turn trough is placed on carries out electricity in the ice-water bath and change, electric current: constant current 85mA, 2h gets final product.From groove, take out film with tweezers after shifting completion, rinsing film in distilled water immerses the methyl alcohol several seconds then, puts into staining fluid (0.1% Xylene Brilliant Cyanine G G-250,40% methyl alcohol, 1% acetate) dyeing 30-60s.Placing then decolours in the destainer (50% methanol aqueous solution) does not have blue look to background and gets final product.With distilled water rinsing film, put film in the filter paper seasoning.Cut the purpose band, send order-checking.
With the aminoacid sequence that records BLAST in ncbi database, find out the proteic gene the highest with its consistence.Utilize the highest gene design primer of homology, extract subtilis Zj016 genome and with it as template, clone's goal gene.
Upstream primer P1 is shown in SEQ ID NO.2; Downstream primer P2 is shown in SEQ ID NO.3.
The PCR system is: each 1 μ L of 10 μ M primer P1 and P2, and 2mM dNTPS 5 μ L, 10 * KOD-Plus-Neo Buffer5 μ L, the KOD-Plus-Neo archaeal dna polymerase 1 μ L of 1U/ μ L, template 0.5 μ L adds distilled water polishing 50 μ L.PCR condition: 94 ℃ of preparatory sex change 5min; 94 ℃ of sex change 30s, 58 ℃ of annealing 30s, 72 ℃ are extended 1min 30s, 30 circulations.PCR product and carrier PUC19 are carried out double digestion respectively and cut the glue recovery; Spend the night in 16 ℃ of connections; Transformed E .coli JM109; At 3 transformants of the dull and stereotyped picking of the LB that is containing ammonia benzyl (100 μ g/mL) resistance, extract recombinant plasmid and go forward side by side and serve Hai Shenggong after performing PCR and the double digestion checking and check order, recombinant plasmid is PUC19-SAP.
According to the dna sequence analysis that records, goal gene encoding mature aminopeptidase and one are contained 31 amino acid whose signal peptides, design another to primer, are template with recombinant plasmid PUC19-SAP, and the clone does not contain the goal gene of signal peptide, and PCR system and condition are the same.
Upstream primer P3 is shown in SEQ ID NO.2; Downstream primer P4 is shown in SEQ ID NO.2.
PCR product and empty carrier pET-28a (+) are carried out double digestion respectively and cut the glue recovery; Spend the night in 16 ℃ of connections; Transformed E .coli JM109; At the dull and stereotyped picking transformant of the LB that is containing that (50 μ g/mL) resistance of card, extract the recombinant plasmid performing PCR of going forward side by side and check order with double digestion checking back, recombinant plasmid is pET-28a (+)-AP.With positive colony plasmid Transformed E .coli BL21 (DE3), do the expression checking at the dull and stereotyped picking list of the LB that contains that (50 μ g/mL) resistance of card bacterium colony.
Embodiment 2:
Picking list bacterium colony is in 5mL LB substratum (blocking that 50 μ g/mL), and 37 ℃ of 200rpm incubated overnight are forwarded to by 1% inoculum size that 37 ℃ of 200rpm are cultured to OD value about 0.8 in the 50mL TB substratum (blocking that 50 μ g/mL) again; Add IPTG to final concentration 0.4mM; The centrifugal 5min of 8000rpm collected thalline after 28 ℃ of 200rpm cultivated 12h, used the Tris-HCl damping fluid suspension thalline of 10mL50mM pH 8.5 again, ultrasonication; Centrifugal collection supernatant carries out the alive and SDS-PAGE checking of enzyme.Record the enzyme 4.8U/mL of being alive of fermented liquid.Control group does not add IPTG induces, and all the other conditions are the same.SDS-PAGE result sees Fig. 5.
Claims (4)
1. a recombination bacillus coli of producing aminopeptidase is that the encoding mature aminopeptidase gene that derives from subtilis Zj016 is imported the genetic engineering bacterium that E.coli BL21 (DE3) obtains.
2. genetic engineering bacterium according to claim 1 is characterized in that said encoding mature aminopeptidase gene is an aminopeptidase gene of having removed 31 amino acid whose signal peptides, and nucleotide sequence is shown in SEQ ID NO.1.
3. the construction process of the recombination bacillus coli of the said production aminopeptidase of claim 1 is characterized in that step is following:
1) collects the pure enzyme of subtilis Zj016 excretory and carry out N terminal amino acid sequence mensuration;
2) aminoacid sequence that records is compared in ncbi database; According to the nucleotide sequence of the protein-high of its consistence and design primer P1 and P2 clone aminopeptidase gene; With said aminopeptidase gene transformation E.coli JM109, obtain recombinant plasmid PUC19-SAP;
3) analyze the aminopeptidase nucleotide sequence, design primer P3 and P4 clone the gene of the encoding mature aminopeptidase that does not contain signal peptide; The gene of said ripe aminopeptidase is connected with carrier pET-28a (+), selects positive plasmid Transformed E .coli BL21 (DE3).
4. like the said method of claim 3, it is characterized in that concrete steps are following:
1) the pure enzyme of subtilis Zj016 excretory is carried out the SDS-PAGE electrophoresis after, take off gel from sheet glass, remove all concentrated glue, gel is immersed electricity changes 30min in the damping fluid; Filter paper soaks 1min in electricity commentaries on classics damping fluid; Pvdf membrane is put in 5min in the distilled water with film after in methyl alcohol, soaking 15s, is put in balance 10min in the transfering buffering liquid; Open the transfer clip then, black orifice plate is put into clip with foam pad, filter paper, gel, film, filter paper, foam pad down in order; Transfer is folded up in the transfer groove, the negative pole of black orifice plate face back up pad, white orifice plate is aimed at the positive pole of back up pad; In transfer groove, add the whole submergences of appropriate amount of buffer solution to transfer groove; Electric turn trough is placed on carries out electricity in the ice-water bath and change electric current: constant current 85mA, 2h gets final product; From groove, take out film with tweezers after shift accomplishing, rinsing film in distilled water immerses the methyl alcohol several seconds then, puts into the staining fluid 30-60s that dyes; Placing then decolours in the destainer to background does not have blue look; With distilled water rinsing film, put film in the filter paper seasoning, cut the purpose band, send order-checking;
2) with the aminoacid sequence that records BLAST in ncbi database, find out the proteic gene the highest with its consistence; Utilize the highest gene design primer of homology, extract subtilis Zj016 genome and with it as template, clone's goal gene; The PCR system is: each 1 μ L of 10 μ M primer P1 and P2, and 2mM dNTPS 5 μ L, 10 * KOD-Plus-NeoBuffer, 5 μ L, the KOD-Plus-Neo archaeal dna polymerase 1 μ L of 1U/ μ L, template 0.5 μ L adds distilled water polishing 50 μ L; PCR condition: 94 ℃ of preparatory sex change 5min; 94 ℃ of sex change 30s, 58 ℃ of annealing 30s, 72 ℃ are extended 1min 30s, 30 circulations; PCR product and carrier PUC19 are carried out double digestion respectively and cut the glue recovery; Spend the night in 16 ℃ of connections; Transformed E .coliJM109; At 3 transformants of the dull and stereotyped picking of the LB that is containing ammonia benzyl resistance, extract recombinant plasmid and go forward side by side and serve Hai Shenggong after performing PCR and the double digestion checking and check order, recombinant plasmid is PUC19-SAP;
3) according to the dna sequence analysis that records; Goal gene encoding mature aminopeptidase and one are contained 31 amino acid whose signal peptides, design another to primer, are template with recombinant plasmid PUC19-SAP; The clone does not contain the goal gene of signal peptide, and PCR system and condition are the same; PCR product and empty carrier pET-28a (+) are carried out double digestion respectively and cut the glue recovery; Spend the night in 16 ℃ of connections; Transformed E .coli JM109; At the dull and stereotyped picking transformant of the LB that is containing that resistance of card, extract the recombinant plasmid performing PCR of going forward side by side and check order with double digestion checking back, recombinant plasmid is pET-28a (+)-AP; With positive colony plasmid Transformed E .coliBL21 (DE3), do the expression checking at the dull and stereotyped picking list of the LB that contains that resistance of card bacterium colony.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103695361A (en) * | 2013-12-06 | 2014-04-02 | 江南大学 | Genetically engineered bacteria for producing proline aminopeptidase and construction method thereof |
| CN110699341A (en) * | 2019-10-08 | 2020-01-17 | 浙江农林大学 | Microbial aminopeptidase and preparation and application thereof |
| CN110699340A (en) * | 2019-10-08 | 2020-01-17 | 浙江农林大学 | Recombinant aminopeptidase T derived from Listeria monocytogenes and application thereof |
| CN113584005A (en) * | 2021-08-27 | 2021-11-02 | 江南大学 | Preparation of aminopeptidase and application of aminopeptidase in protein debittering |
| CN115261296A (en) * | 2022-03-15 | 2022-11-01 | 西南大学 | Recombinant escherichia coli for expressing aminopeptidase M1AP and construction method |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110699340A (en) * | 2019-10-08 | 2020-01-17 | 浙江农林大学 | Recombinant aminopeptidase T derived from Listeria monocytogenes and application thereof |
| CN113584005A (en) * | 2021-08-27 | 2021-11-02 | 江南大学 | Preparation of aminopeptidase and application of aminopeptidase in protein debittering |
| CN115261296A (en) * | 2022-03-15 | 2022-11-01 | 西南大学 | Recombinant escherichia coli for expressing aminopeptidase M1AP and construction method |
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Application publication date: 20120613 |