CN101386868A - Method for improving expression level of recombinant protein in kluyveromyces - Google Patents
Method for improving expression level of recombinant protein in kluyveromyces Download PDFInfo
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- CN101386868A CN101386868A CNA2008102003441A CN200810200344A CN101386868A CN 101386868 A CN101386868 A CN 101386868A CN A2008102003441 A CNA2008102003441 A CN A2008102003441A CN 200810200344 A CN200810200344 A CN 200810200344A CN 101386868 A CN101386868 A CN 101386868A
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Abstract
The invention belongs to the technical field of gene engineering, in particular to a technical proposal used to improve the expression level of recombinant protein. In the method, a promoter, alpha-signal peptide, target protein of an inulase gene are orderly connected with a terminal subcode sequence of the inulase gene first; then the sequence is inserted into a Kluyveromyces lactis expression vector; finally the Kluyveromyces lactis is transformed and fermented, and a fermented supernatant is taken and separated. The method can accelerate the remarkable growth of the yield of the recombinant protein expressed in the Kluyveromyces lactis. The technical proposal can be used to improve the expression amount of various proteins and provide a new method for reducing production cost and enlarging production scale.
Description
Technical field
The invention belongs to gene engineering technology field, be specifically related to a kind of technical scheme that improves the expression of recombinant proteins level.
Background technology
Yeast is the single celled lower eukaryotes of a class, has protokaryon and eukaryotic characteristics concurrently: both cultivated conveniently, breeding is fast, cost is low, is easy to genetic manipulation, has Secretory Pathway again, can translate post-treatment to the eukaryotic gene product and modify, obtain correctly folding, activated protein.Therefore yeast expression system is described as the expression system (F.R.Schmidt that commercial value is arranged most, Recombinant expression systems in thepharmaceutical industry, Appl Microbiol Biotechnol, 65:363-372,2004).Except that widely used yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) and pichia pastoris phaff (Pichia pastoris) expression system, also having a class is the unconventional yeast expression system of representative with kluyveromyces (Kluyveromyces).
Kluyveromyces mainly comprises Kluyveromyces lactis (Kluyveromyces lactis), Kluyveromyces fragilis (Kluyveromyces fragilis), this kluyveromyces of equine kinds such as (Kluyveromyces marxianus), wherein Kluyveromyces lactis be a kind of can be with the yeast of lactic acid as unique charcoal source growth, it is extremely simple to have nutritional requirement, it is vigorous to grow, biomass is big, growth temperature wide accommodation (25 ℃-46 ℃), the secretory protein ability is strong, to human security (GRAS, Generally Regarded as Safe) characteristics such as, it is a kind of comparatively ideal expression of recombinant proteins host cell, set up the more perfect Kluyveromyces lactis expression system that is suitable for exogenous gene expression so far, utilize the people of this system successful expression human serum albumin, alpha-galactosidase, multiple recombinant protein (Albert J.J.van Ooyen et al such as ox rennin, Heterologousprotein production in the yeast Kluyveromyces lactis, FEMS Yeast Res 6:381-392,2006).
Yeast saccharomyces cerevisiae HAP1 gene (ScHAP1) a kind of transcription factor of encoding, regulate and control the transcriptional expression (Pfeifer of some cellular respiration pathways metabolism indispensable genes, K., Kim, K.S., Kogan, S.and Guarente, L.1989.Functional dissection and sequence of yeast HAPI activator.Cell.56:291-301).As the ScHAP1 homologous gene, the function of Kluyveromyces lactis HAP1 gene (K1HAP1) it be not immediately clear.
Summary of the invention
The object of the present invention is to provide a kind of novel method that improves recombinant protein expression level in kluyveromyces.
The invention provides a kind of method that improves recombinant protein expression amount in kluyveromyces, this method comprises the steps:
(1) the terminator encoding sequence with promotor, α-signal peptide, target protein and the inulinase gene of inulinase gene is connected successively; (promotor of inulinase gene and terminator sequence are seen the GenBank number of logining: AF178979; The GenBank number of logining of α-signal peptide: J01340)
(2) sequence that (1) is obtained is inserted kluyveromyces lactis expression vector pUKD-S or pUKD;
(3) recombinant vectors that obtains with (2) transforms kluyveromyces, and fermentation is got the fermented supernatant fluid separation and got final product.
Among the present invention, the kluyveromyces that adopts in (3) can be Kluyveromyces lactis, chick pea kluyveromyces, this kluyveromyces of equine or Kluyveromyces fragilis.
Among the present invention, used Kluyveromyces lactis can be a MW179-1D HAP1 genetic flaw bacterial strain.Lactic acid Crewe dimension bacterial strain MW179-1D information is seen document: Imrichova D et al.YAP1-mediatedKNQ1 expression in Kluyveromyces lactis, FEMS Yeast Research, 5 (4-5): 323-329,2005.
Among the present invention, (1) promotor can be by (Zhang J et al.Cloning of the Kc URA3 Gene and Development of aTransformation System for Kluyveromyces cicerisporus is seen in the source with chick pea kluyveromyces Y179 in, Appl MicrobiolBiotechnol., 62 (4): 387-391, .2003.) total DNA is a template, with P1 and P2 is primer, and pcr amplification obtains; The sequence of P1 is shown in SEQ ID NO 2, and the sequence of P2 is shown in SEQ ID NO 3.
Among the present invention, terminator can be to be template by the total DNA with chick pea kluyveromyces Y179 in (1), is primer with P5 and P6, and pcr amplification obtains; The sequence of P5 is shown in SEQ ID NO 6, and the sequence of P6 is shown in SEQ ID NO 7.
Among the present invention, the target protein in (1) can be enzyme, cytokine, polypeptide, antibody or vaccine antigen.Recombinant protein among the present invention can be commercial useful protein, comprises enzyme, cytokine, polypeptide, antibody, vaccine antigen, and this proteinic gene of encoding can be from microorganism, plant, animal or human.
In one embodiment of the present of invention, the target protein in (1) can be an inulinase.
Among the present invention, α-signal peptide and inulinase encoding sequence are template by the total DNA with chick pea kluyveromyces Y179, are primer with P3 and P4, and pcr amplification obtains; The sequence of P3 is shown in SEQ ID NO4, and the sequence of P4 is shown in SEQ ID NO 5.
Inulinase (inulinase) is a class hydrolysis β-2, a class lytic enzyme of 1-D-polyfructosan fructose glycosidic link.The mode that inulinase is cut the polyfructosan sugar chain according to its enzyme is divided into excision enzyme (EC3.2.1.80) and restriction endonuclease (EC3.2.1.7).The microorganism that produces inulinase mainly is a fungi.The chick pea kluyveromyces inulinase gene GenBank number of logining is AF178979, and promotor and terminator sequence are included.Inulinase has important application prospects in the following areas.The firstth, the preparation of high fructose syrup.High fructose syrup is the ideal natural sweeteners, and the sweetness ratio sucrose of fructose exceeds 80%; Can be fit to diabetes patient.Also have nutrient health-care function simultaneously, can regulate stomach, improve immunizing power, toxin-expelling and face nourishing, degraded blood sugar and superior physiologically active such as anticancer, be called as third generation functional food.Second aspect is the application in bioenergy field.At present, the production of bio-ethanol mainly is to be raw material with the W-Gum, and the plantation of raw material faces the embarrassment with human grain contention soil.The main component inulin of jerusalem artichoke, its growth conditions is slightly mad, and plantation does not occupy cultivated land, the saltings of can not only growing, can also be grown in desert area, can prevent desertification of land, can become replenishing of present starch ethanol and back Preparation Method for the raw material production bio-ethanol with the inulin.Inulin is hydrolyzed into monose under the effect of inulinase, can directly be utilized by yeast, produces ethanol.Therefore, whether feasible key is the height of cost to preparation inulin ethanol, and this wherein produces the inulinase cost is key in the key.
Method of the present invention specifies as follows:
1. the recombinant protein kluyveromyces expression plasmid of inulinase gene promoter, terminator control makes up.
(1) the recombinant protein expression casette makes up
With the kluyveromyces genomic dna is template, the design Auele Specific Primer, utilize round pcr increase respectively circumscribed inulinase gene promoter and terminator sequence, again by pcr amplification recombinant protein gene order, with the inulinase gene promoter, terminator and recombinant protein gene order are carried out digestion with restriction enzyme respectively and are handled, ligation through dna ligase, with the inulinase gene promoter, recombinant protein gene and inulinase gene terminator sequence are cloned successively on the pBS_SK plasmid, obtain by the inulinase gene promoter, the recombinant protein expression casette that three kinds of elements of recombinant protein gene and inulinase gene terminator sequence constitute.Genetic engineering technique methods such as PCR, the enzyme that adopts cut, connection, intestinal bacteria conversion are ordinary method, with reference to " molecular cloning experiment guide " (J.Sambrook, et al., second edition, 1996).
(2) recombinant protein kluyveromyces expression plasmid makes up
PUKD-S is at the stable carrier of kluyveromyces camber, is made of kluyveromyces plasmid pKD1, chick pea kluyveromyces KcURA3 gene and intestinal bacteria pUC19 sequence.Carrier pUKD-S is cut with restriction enzyme Sse3871, fill and lead up sticky end with the Klenow enzyme; Expression of recombinant proteins box on the clone PBS-SK plasmid is downcut with restriction enzyme, and the expression cassette fragment is is also filled and led up sticky end with the Klenow enzyme after separating.Then, the expression cassette fragment is connected with dna ligase with carrier pUKD-S fragment, obtains the recombinant protein gene kluyveromyces expression plasmid of inulinase gene promoter and terminator control.
2. the recombinant protein kluyveromyces expression plasmid of inulinase gene promoter, terminator control imports HAP1 gene defection type kluyveromyces yeast strains, makes up the expression of recombinant proteins engineering bacteria.
The recombinant protein gene kluyveromyces expression plasmid of inulinase gene promoter and terminator control is imported the kluyveromyces yeast strains (Ura3 of HAP1 genetic flaw with LiAc intact cell method for transformation
-), the yeast that screening can be grown in lacking the selective medium of uridylic promptly obtains recombinant protein kluyveromyces engineering strain.Yeast cell LiAc method for transformation is with reference to " yeast genetics method experiment guide " (A.Adams, et al., 2000).
On the other hand, the present invention also provides a kind of carrier of recombinant protein at the kluyveromyces expression amount that be used for improving, and this carrier is made up of the terminator encoding sequence and the carrier sequence of the promotor of inulinase gene, α-signal peptide, target protein, inulinase gene; Wherein the terminator encoding sequence of the promotor of inulinase gene, α-signal peptide, target protein, inulinase gene connects successively, carrier is pBS_SK, pUKD-S or pUKD, and the dna fragmentation that the terminator encoding sequence of the promotor of inulinase gene, α-signal peptide, target protein, inulinase gene is formed inserts the multiple clone site of carrier.
The present invention has made up the kluyveromyces expression plasmid that contains inulinase gene promoter, terminator element and recombinant protein gene order, and with in this plasmid importing kluyveromyces yeast strains, build up the kluyveromyces genetic engineering bacterium and produce recombinant protein, obviously promote the kluyveromyces express recombinant protein.The present invention can be used for improving the expression amount of multiple protein, and new method is provided for reducing production costs, expanding the scale of production.
Description of drawings
Fig. 1: inulinase gene expression plasmid pUKD-S-α F-PinuT structural representation.
Fig. 2: Kluyveromyces lactis engineering bacteria K1-WT/pUKD-S-α F-PinuT fermentation secreting, expressing inulinase polyacrylamide gel electrophoresis figure.
Fig. 3: Kluyveromyces lactis engineering bacteria klhap1 Δ/pUKD-S-α F-PinuT fermentation secreting, expressing inulinase polyacrylamide gel electrophoresis figure.Compare with Fig. 2, the concentration of inulinase protein band is deepened greatly, illustrates that its expression output increases greatly.
Fig. 4: Kluyveromyces lactis engineering bacteria K1-WT/pUKD-S-α F-PinuT and klhap1 Δ/pUKD-S-α F-PinuT fermented sample unit volume inulinase activation analysis.Wherein, what represent with the black triangle is Kluyveromyces lactis engineering bacteria K1-WT/pUKD-S-α F-PinuT fermented sample, and what represent with the black square is klhap1 Δ/pUKD-S-α F-PinuT fermented sample.
Fig. 5: Kluyveromyces lactis engineering bacteria K1-WT/pUKD-S-α F-PinuT and klhap1 Δ/pUKD-S-α F-PinuT fermented sample unit thalline is produced the inulinase activation analysis.
Embodiment
1.. chick pea kluyveromyces inulinase expression casette makes up
At first, total DNA with chick pea kluyveromyces Y179 is a template, be primer with P1 and P2, P5 and P6 respectively, amplify promoter region dna fragmentation, the inulinase gene coding region of the circumscribed inulinase gene of chick pea kluyveromyces and stop the subarea dna fragmentation by round pcr, be template with the pPIC9K plasmid again, with P3 and P4 is primer, amplifies α-Factor signal peptide dna fragmentation by round pcr.Restriction enzyme site according to each fragment design carries out corresponding restriction enzyme processing, 3 endonuclease bamhis of gained are connected with the plasmid pBS_SK that handles through restriction enzyme EcoR1, HindIII double digestion, obtain plasmid pBS_SK-α F-PinuT, promptly the inulinase expression casette that is made of inulinase gene promoter, α-Factor signal peptide, inulinase gene coding region and terminator is cloned on the plasmid pBS_SK.
The nucleotide sequence of 6 used primers is as follows:
Two primer sequences of clone's inulinase gene promoter fragment are:
P1:5 ' AG
GAATTCAAAACGACAAGAGAAGCAAACACA3 ' (see SEQ IDNO 2, underscore partly is the BamH1 restriction enzyme site)
P2:5 ' AC
ACTAGTATCTAACAAAAAAAAAATTAAATGTGT3 ' (see SEQID NO 3, underscore partly is the Spel1 restriction enzyme site)
Clone's α-two primer sequences of Factor fragment are:
P3:5 ' AC
ACTAGTTGAGATTTCCTTCAATTTTTACTGCA3 ' (see SEQ IDNO 4, underscore partly is the Spel1 restriction enzyme site)
P4:5 ' AC
AGATCTTCTTTTATCCAAAGATACCCCTTCTTC 3 ' (see SEQID NO 5, underscore partly is the BglII restriction enzyme site)
Clone's inulinase gene coding region with two primer sequences of termination subarea fragment is:
P5:5 ' AC
AGATCGATGGTGACAGCAAGGCCATCAC3 ' (see SEQ ID NO6, underscore partly is a Bgl II restriction enzyme site)
P6:5 ' AT
AAGCTTTAGGCAGGGCTGTGATACACCTGT3 ' (see SEQ IDNO 7, underscore partly is the HindIII restriction enzyme site)
2. chick pea kluyveromyces inulinase gene expression plasmid pUKD-S-α F-PinuT makes up
1. middle plasmid pBS_SK-α F-PinuT is cut out inulinase expression casette dna fragmentation with restriction enzyme BamH1, HindIII, and fragment is separated the back and is filled and led up sticky end with the Klenow enzyme; Yeast vector pUKD-S is cut with restriction enzyme Sse3871, also fill and lead up sticky end with the Klenow enzyme.Both connect with dna ligase then, obtain chick pea kluyveromyces inulinase gene expression plasmid pUKD-S-α F-PinuT, and collection of illustrative plates is seen Fig. 1.
Kluyveromyces lactis engineering bacteria K1-WT/pUKD-S-α F-PinuT and engineering bacteria klhap1 Δ/pUKD-S-α F-PinuT that embodiment 2 expresses the inulinase gene make up.
Plasmid pUKD-S-α F-PinuT transforms Kluyveromyces lactis MW179-1D bacterial strain and HAP1 genetic flaw bacterial strain MW179-1D hap1 Δ respectively, adopts the LiAc conversion method.Kluyveromyces lactis HAP1 gene order is SEQ1.ID.NO 1.
1. inoculate single bacterium colony in the 3ml liquid YEPD substratum 30 ℃ shaking culture 16-18 hour.
2. culture is diluted among the 50ml liquid YEPD, makes initial OD
600=0.2, put OD 30 ℃ of shaking culture 3-5 hours
600=0.4-0.6.
3. 5K, 5 minutes centrifugal collecting cells.
4. abandon nutrient solution, with 25ml sterilized water suspension cell, the same more centrifugal.
5. abandon water,, transfer in the aseptic 1.5ml centrifuge tube with 1ml0.1mol/L LiAc suspension cell.
6. high speed centrifugation sedimentation cell in 5 second is removed supernatant, adds the abundant suspension cell of 500 μ l 0.1mol/L LiAc then.
7. get 50 μ l cells and be added in the centrifuge tube of mark, high speed centrifugation sedimentation cell in 5 second is removed supernatant, then successively
Add 240 μ l PEG (50%w/v), 36 μ l 1.0mol/L LiAc, 10 μ l ssDNA (5mg/ml), 50 μ l water and plasmid DNA (0.1-1 μ g).
8. abundant mixing cell is put 30 ℃ of insulations 30 minutes, puts 42 ℃ of water-bath heat shocks 20 minutes then.
9. high speed centrifugation is 15 seconds, removes supernatant, adds 150 μ l sterilized water suspension cells, is coated with selectivity SD culture medium flat plate then, puts 30 ℃ of cultivations.Selectivity SD medium component is: 0.67% YNB, 2% glucose, 0.002% uridylic, 0.002% methionine(Met) and 2% agar.
1. Kluyveromyces lactis engineering bacteria K1-WT/pUKD-S-α F-PinuT and klhap1 Δ/pUKD-S-α F-PinuT are inserted respectively in the 3ml selectivity SD liquid nutrient medium, at 30 ℃, in the 250rpm rotating speed shaking table after the overnight incubation as seed liquor.Selectivity SD liquid culture based component is: 0.67%YNB, 2% glucose, 0.002% uridylic and 0.002% methionine(Met).
2. seed liquor is equipped with in the 150ml triangular flask of 25ml liquid YEPD substratum with the ratio access of 1:25,, cultivated 120 hours in the 250rpm rotating speed shaking table at 30 ℃.Liquid YEPD medium component is: 1% yeast extract, 2% peptone and 2% glucose.
3. drew fermentation broth sample every 24 hours, the centrifugal 5min precipitation of 5000rpm thalline is got supernatant liquor and is carried out polyacrylamide gel electrophoresis analysis of expression product inulinase and inulinase activation analysis.
4. Fig. 2 and Fig. 3 are in 120 hours fermentation periods, different time points fermented sample polyacrylamide gel electrophoresis result, compare with Kluyveromyces lactis engineering bacteria K1-WT/pUKD-S-α F-PinuT, klhap1 Δ/pUKD-S-α F-PinuT strain enzyme-producing amount obviously improves.
5. fermented liquid inulinase determination of activity result shows (Fig. 4, Fig. 5): no matter the unit volume enzyme is lived, still the unit bacterial enzyme is lived, HAP1 genetic flaw strain klhap1 Δ/pUKD-S-α F-PinuT is all apparently higher than non-defective strain K1-WT/pUKD-S-α F-PinuT, ferment 120 hours the time, HAP1 genetic flaw strain klhap1 Δ/pUKD-S-α F-PinuT yield of enzyme reaches 391.2 units per ml, but not defective strain K1-WT/pUKD-S-α F-PinuT yield of enzyme has only 178.2 units per ml, and HAP1 genetic flaw strain product inulinase amount is 2.2 times of non-defective strain.
Sequence table
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＜120〉a kind of method that improves recombinant protein expression amount in kluyveromyces
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Claims (9)
1. a method that improves recombinant protein expression amount in kluyveromyces is characterized in that, this method comprises the steps:
(1) the terminator encoding sequence with promotor, α-signal peptide, target protein and the inulinase gene of inulinase gene is connected successively;
(2) sequence that (1) is obtained is inserted kluyveromyces lactis expression vector pUKD-S or pUKD;
(3) recombinant vectors that obtains with (2) transforms kluyveromyces, and fermentation is got the fermented supernatant fluid separation and got final product.
2. the method for claim 1 is characterized in that, the kluyveromyces that adopts in (3) is Kluyveromyces lactis, chick pea kluyveromyces, this kluyveromyces of equine or Kluyveromyces fragilis.
3. method as claimed in claim 2 is characterized in that, used Kluyveromyces lactis is a MW179-1D HAP1 genetic flaw bacterial strain.
4. the method for claim 1 is characterized in that, promotor is to be template by the total DNA with chick pea kluyveromyces Y179 in (1), is primer with P1 and P2, and pcr amplification obtains; The sequence of P1 is shown in SEQ ID NO2, and the sequence of P2 is shown in SEQ ID NO3.
5. the method for claim 1 is characterized in that, terminator is to be template by the total DNA with chick pea kluyveromyces Y179 in (1), is primer with P5 and P6, and pcr amplification obtains; The sequence of P5 is shown in SEQ ID NO6, and the sequence of P6 is shown in SEQ ID NO7.
6. the method for claim 1 is characterized in that, the target protein in (1) is enzyme, cytokine, polypeptide, antibody or vaccine antigen.
7. the method for claim 1 is characterized in that, the target protein in (1) is an inulinase.
8. method as claimed in claim 7 is characterized in that, α-signal peptide and inulinase encoding sequence are template by the total DNA with chick pea kluyveromyces Y179, is primer with P3 and P4, and pcr amplification obtains; The sequence of P3 is shown in SEQ ID NO4, and the sequence of P4 is shown in SEQ ID NO5.
9. one kind is used for improving the carrier of recombinant protein at the kluyveromyces expression amount, it is characterized in that, this carrier is made up of the terminator encoding sequence and the carrier sequence of the promotor of inulinase gene, α-signal peptide, target protein, inulinase gene; Wherein, the terminator encoding sequence of the promotor of inulinase gene, α-signal peptide, target protein, inulinase gene connects successively; Carrier is pBS_SK, pUKD-S or pUKD; The dna fragmentation that the terminator encoding sequence of the promotor of inulinase gene, α-signal peptide, target protein, inulinase gene is formed inserts the multiple clone site of carrier.
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| CN105524923A (en) * | 2015-12-28 | 2016-04-27 | 大连理工大学 | Kluyveromyces marxianus derived high-expression promoter and expression system thereof |
| WO2017036294A1 (en) * | 2015-08-28 | 2017-03-09 | 复旦大学 | Kluyveromyces marxianus and use thereof |
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| CN114341358A (en) * | 2019-07-25 | 2022-04-12 | 科学与工业研究委员会 | Recombinant vector for high expression of protein in yeast |
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| CN105524923A (en) * | 2015-12-28 | 2016-04-27 | 大连理工大学 | Kluyveromyces marxianus derived high-expression promoter and expression system thereof |
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| CN107102050A (en) * | 2017-05-16 | 2017-08-29 | 肖玲君 | A kind of method for the expression for quantitatively determining the destination protein expressed in microorganism |
| CN113528574A (en) * | 2018-08-07 | 2021-10-22 | 康码（上海）生物科技有限公司 | Signal peptide related sequence and application thereof in protein synthesis |
| CN113481226A (en) * | 2018-08-07 | 2021-10-08 | 康码（上海）生物科技有限公司 | Signal peptide related sequence and application thereof in protein synthesis |
| CN113528575A (en) * | 2018-08-07 | 2021-10-22 | 康码（上海）生物科技有限公司 | Signal peptide related sequence and application thereof in protein synthesis |
| CN113528574B (en) * | 2018-08-07 | 2022-06-21 | 康码（上海）生物科技有限公司 | Signal peptide related sequence and application thereof in protein synthesis |
| CN113528575B (en) * | 2018-08-07 | 2022-06-21 | 康码（上海）生物科技有限公司 | Signal peptide related sequence and application thereof in protein synthesis |
| CN113481226B (en) * | 2018-08-07 | 2022-06-21 | 康码（上海）生物科技有限公司 | Signal peptide related sequence and application thereof in protein synthesis |
| CN114341358A (en) * | 2019-07-25 | 2022-04-12 | 科学与工业研究委员会 | Recombinant vector for high expression of protein in yeast |
| CN114807180A (en) * | 2022-03-10 | 2022-07-29 | 上海玉华生命科技发展有限公司 | Process for expressing human superoxide dismutase by using Kluyveromyces fragilis |
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