CN105969747A - Method for secreting and producing human-derived core fucose-base transferases by aid of pichia pastoris expression systems - Google Patents
Method for secreting and producing human-derived core fucose-base transferases by aid of pichia pastoris expression systems Download PDFInfo
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Abstract
The invention provides a method for producing, efficiently secreting and expressing core fucose-base transferases FUT8 in pichia pastoris expression systems, and belongs to the field of genetic engineering and the field of protein engineering. The method mainly comprises constructing the pichia pastoris secretion and expression systems for the recombinant human-derived core fucose-base transferases FUT8; separating and purifying the recombinant human-derived core fucose-base transferases FUT8; detecting the enzymatic activity of the recombinant human-derived core fucose-base transferases FUT8. The method has the advantages that the core fucose-base transferases FUT8 synthesized, secreted and expressed by the aid of the method can be widely applied to protein inhibitor, biological function and in-vitro oligosaccharide research; the method is low in synthesis cost, short in synthesis period and easy to technically implement, and the core fucose-base transferases FUT8 obtained by the aid of the method are high in purity.
Description
Technical field
The present invention relates to oligosaccharide biosynthesis, particularly one utilizes pichia yeast expression system secretion to produce recombination human source
The method of core fucosyltransferase FUT8, belongs to genetic engineering field and protein engineering field.
Background technology
The memebrane protein with two membrane-spanning domains that major part glycosyl transferase is assembled on endoplasmic reticulum and Golgi membrane,
Each monosaccharide is catalyzed on corresponding sugar chain formation in endoplasmic reticulum and Golgi body and has bioactive by glycosyl transferase
Sugar chain.
Up to now, it is known that fucosyltransferase have 11 kinds, core rock algae in fucosyl transferase gene family
Glycosyl transferase Fut8 is the most ancient gene.GDP-L-fucose: N-acetyl-β-D-Glucose amine core fucose group-transfer
Enzyme (Fut8) is GDP-fucose (GDP-Fuc) is transferred to mixed type or complexity N glycan core structures interior
6 carbon atoms of side 2-Acetamido-2-deoxy-D-glucose (GlcNAc), form α 1,6 glycosidic bond and connect the glycosyl of core fucose structure
Transferring enzyme, therefore also known as α 1,6 fucosyltransferases, Fut8 is primarily targeted for middle part Golgi body, the core of catalytic substrate albumen
Fucosylation is modified.
Fut8 is the unique glycosyl transferase modifying core fucosylation, and its catalytic reaction is as shown in Figure 5, it is possible to adjust
The joint space structure of protein and biological function, as intermolecular interaction, cell and intercellular be mutually distinguishable, protein
Be properly positioned, cellular signal transduction etc..Fut8-/-Mice has emphysema Phenotype, this is because TGF-1
(TGF1) the core fucose base defect on receptor then reduces the binding ability of TGF-1 (TGF1) and receptor, activates
The expression of matrix metalloproteinase (MMP), causes emphysema pathological changes；Fut8 gene knockout causes the blood vessel endothelium life of cell surface
Growth factor receptor body expression declines.It addition, Stubbs etc. find that core fucosylation degree can affect on glycoprotein molecule
The molecular conformation of N-sugar chain and flexibility.
Core fucosyltransferase is expressed in a lot of cancer patient's bodies or activity all has rising, a large amount of acquisition core rocks
Algae glycosyl transferase is the premise of research core fucosyltransferase function, and investigation finds only the thinnest in COS-1 suckling
Expressing the core fucosyltransferase in remarkable source in born of the same parents, the synthesis of people source core fucosyltransferase is repaiied for external oligosaccharide
Adorn significant, active core fucosyltransferase can be synthesized in mammalian cell but its cycle long cost
Many.
Pichia yeast expression system is the most successful efficient expression system, and its genetic background is the clearest, external source
Gene can stablize heredity, expresses yield high, and low cost is particularly suitable for expressing activated albumen.It addition, as eukaryotic expression system
System, Pichia sp. has the post translational modification function of other animal and plant cellss similar, protein folding etc..Due to Pichia sp. table
The system of reaching has the advantage of protokaryon and eukaryotic expression system concurrently, obtains increasingly extensive application, with this be in genetic engineering field
System expresses core fucosyltransferase can be as preferably selecting approach.But yet there are no core fucosyltransferase so far
The report that successful expression synthesizes in a large number in Pichia sp..
Summary of the invention
For above-mentioned situation, for overcoming the defect of prior art, the object of the invention is just to provide one and utilizes Pichia sp.
Expression system produces the method for Golgi membrane protein core fucosyltransferase, can effectively solve core fucose base and turn
Move enzyme and prepare problem.
The technical scheme that the present invention solves is to include three parts: recombination human source core fucosyltransferase FUT8 finishes red
The structure of yeast secreted expression system, the isolated and purified and recombination human source core of recombination human source core fucosyltransferase FUT8
The detection of fucosyltransferase FUT8 enzymatic activity, specifically includes following steps
(1) according to Protein Data Bank (UniProtKB-Q9BYC5) people source core fucosyltransferase structure
Information design fusion protein sequence, utilizes RCSB PDB to announce core fucosyltransferase Protein Information and removes core fucose
The trans-membrane region that based transferase N end is combined with Golgi membrane, and at the N end introducing alpha signal peptide of residue sequence, at C end
Termination codon before introduce 6 His aminoacid, for isolated and purified label, facilitate follow-up protein purification, protein mark
Sign as histidine (6XHis), amino sequence table SEQ ID No.1；
Utilize Pichia sp. codon preference to synthesize core fucosyltransferase FUT8 gene, be not change core
On the basis of fucosyltransferase FUT8 aminoacid sequence, according to Pichia sp. Preference codon, core fucose base is turned
Move enzyme FUT8 complete genome sequence be optimized and synthesize, simultaneously at the 5 of core fucosyltransferase FUT8,End and 3,End introduces
Restriction enzyme site.Its nucleotides sequence list is SEQ ID No.2；
Described Pichia sp. is Pichia yeast cell X-33.
(2) utilizing restricted enzyme to build recombinant expression plasmid pPICZ α A-FUT8, method is: will close in step (1)
The recombination human source core fucosyltransferase FUT8 become and expression plasmid carrier pPICZ α A, utilizes restricted enzyme to carry out
Enzyme action, reclaims purpose fragment, utilizes T4DNA ligase to be connected with plasmid vector X endonuclease bamhi by the endonuclease bamhi of FUT8, obtain
Recombinant expression plasmid pPICZ α A-FUT8, is transformed in escherichia coli TP10 and expands；
Its concrete operation step is: utilize restricted enzyme X hoI and X baI by the core rock of synthesis in step (1)
Algae glycosyltransferase gene FUT8 and inducible expression vector pPICZ α A carries out double digestion, and gel electrophoresis reclaims target after separating
PPICZ α A linear fragment gene after DNA fragmentation i.e. double digestion, then utilizes T4DNA ligase that target DNA fragments is carried out 16
DEG C connecting overnight, 42 DEG C of thermal shocks convert escherichia coli TP10 competent cells, are coated with (bleomycin) Han 25ug/ml Zeocin
LB culture medium flat plate, be placed in overnight incubation at 37 DEG C, random picking monoclonal, extract recombinant expression plasmid pPICZ α A-
FUT8, identifies positive colony by double digestion；
Described LB culture medium is by yeast powder 5g, 10g peptone, 5g sodium chloride, and regulation pH value, to 7.2-7.4, adds water
Make to 1000ml.
(3) restricted enzyme is utilized to convert Pichia sp. after recombinant expression carrier pPICZ α A-FUT8 carries out linearisation
The competent cell of bacterium cell X-33, the application YPDS flat screen containing antibiotic selects induction type positive transformant；
Utilize restricted enzyme PmeI enzyme action inducible expression vector pPICZ α A-FUT8, take 5-20ug plasmid and carry out electricity
Hitting conversion pichia pastoris X-33 competent cell, the parameter of eppendorf-Eporator electricity conversion instrument used is: voltage
1.5KV, electric capacity hinders 200 Ω, and discharge time, 4.8-5.2ms, added 1.0M sorbitol ice-cold for 1.0ml immediately after electric shock, 30 DEG C
The coating YPDS flat board containing 100ug/ml Zeocin after shaken cultivation 1-2h, grows induction after being placed in 28-30 DEG C of cultivation 2-4d
Type positive transformant.
Described YPDS flat board is by 10g yeast powder, 20g peptone, 20g-D glucose, 15g agar, 10g sorbitol
Add water to 1000ml make.
(4) the fermentation screening of recombiant protein FUT8 Pichi strain is expressed: the induction type positive that step (3) filters out turns
When beggar cultivates OD600=2-6 in BMGY culture medium, changing culture medium is the abduction delivering that BMMY carries out recombiant protein, induction
2-4 days, fermented liquid supernatant with SDS-PAGE detection protein expression amount and detected whether as core rock algae by Western blot method
Glycosyl transferase, filters out the Pichia sp. of induction type high-level secretory expression recombination human source core fucosyltransferase FUT8
Recombinant bacterial strain；
Concrete operations are: the induction type positive transformant filtered out from step (3) streak culture 2-3 on YPDS flat board
My god, picking list colony inoculation is in the 5ml centrifuge tube containing 1ml BMGY culture medium, and 30 DEG C of 200rpm cultivate OD600=2-6
Time, changing culture medium is the abduction delivering that BMMY carries out recombiant protein, and in BMMY culture medium culturing bacterial strain, every 24h addition methanol is the denseest
Degree is 0.5%, and cultivates at 30 DEG C of 200rpm, and after induction 2-4d, 5000rpm is centrifugal collects supernatant.Albumen is detected with SDS-PAGE
Expression also detects by Western blot method, and each sample parallel two row detects, and final screening obtains high level also
The Pichi strain of correct excreting and expressing recombinant human source core fucosyltransferase FUT8；
Described BMGY culture medium is: 10g yeast powder, 20g peptone, 10g sorbitol, the 100mM phosphoric acid buffer of pH6.0
Liquid, 13.4g are without aminoacid yeast nitrogen, 4X10-4Biotin, add water to 1000ml and make；
Described BMMY culture medium is: 10g yeast powder, 20g peptone, the 100mM phosphate buffer of pH6.0,13.4g without
Aminoacid yeast nitrogen, 4X10-4Biotin, 5ml methanol, add water to 1000ml and make；
Screened by above-mentioned fermentation, it is thus achieved that there is hypersecretion and express finishing of recombination human source core fucosyltransferase FUT8
Red yeast strain pPICZ α A-FUT8/X-33.Through inspection, the recombination human source core fucosyltransferase FUT8 of expression has sky
So biologic activity.
(5) isolated and purified recombination human source core fucosyltransferase in Pichia sp. recombinant bacterial strain fermented supernatant fluid
FUT8, by step
(4) what screening obtained has the Pichia sp. weight of hypersecretion expression recombination human source core fucosyltransferase FUT8
Group bacterial strain carries out fermentation inducement expresses 2-4 days, 5000rpm, 4 DEG C fermentation liquid is centrifuged 10min, collect supernatant, use Ni-
NTA SepharoseTMExcel column one-step method is purified, it is thus achieved that the recombiant protein FUT8 of purification.
With 10 column volumes of equilibration buffer Ni-NAT chromatographic column, by the protein sample after above-mentioned dialysis equilibrium
Post, carries out affine displacement chromatography, and after sample upper prop, continuation level pad washes 20 column volumes, the most respectively with containing
Have 40,60,80,100,120 and the level pad of 200mM imidazole concentration carry out gradient stepwise elution, collect eluent, i.e. obtain
Obtain recombiant protein FUT8 after purification, by the effect of SDS-PAGE detection protein purification；
Core fucosyltransferase after purification uses the method desalination of desalting column, cryopreservation.
The invention have the benefit that the retention signal that the recombiant protein of expression eliminates on Golgi membrane makes core
Fucosyltransferase successful secretion is in culture medium；Gene order FUT8 is carried out according to the Preference of Pichia sp. codon
Optimize so that it is be more suitable in Pichia sp. expressing；The recombiant protein expressed is with special label, it is easy to carry out recombiant protein
Isolated and purified；By the FUT8 after isolated and purified in Pichia sp. recombinant bacterial strain expression supernatant, there is enzymatic activity, can be direct
Modify for external oligosaccharide.The low cost of synthesis, the cycle of synthesis is short, and in technical operation easily, the purity of gained FUT8 is high.
Accompanying drawing explanation
Fig. 1 is the recombination human source core fucosyltransferase FUT8 gene structure display that the present invention synthesizes；
Fig. 2 is that the expression recombination human source core fucosyltransferase FUT8 inducible plasmid of the structure of the present invention expresses load
The schematic diagram of body pPICZ α A-FUT8；
Fig. 3 is whether the detection core fucosyltransferase of the present invention expresses correct coomassie brilliant blue staining and albumen
Trace schematic diagram, wherein, A figure is coomassie brilliant blue staining: 1) pre-dyed Marker, 2) recombination human source core fucose after purification
Based transferase FUT8, B figure is Western blot: 1) people source core fucosyltransferase FUT8 antibody test recombiant protein, 2) and His
Tag antibody detection recombiant protein；
Fig. 4 is the detection recombination human source core fucosyltransferase FUT8 proteinase activity of the present invention, and wherein, A is the end
Analyte detection schematic diagram, a peak is FUT8 catalytic substrate peak, and appearance time is about 3.7min, B for adding recombination human source core fucose
Producing catalytic reaction schematic diagram after based transferase FUT8, a peak is FUT8 catalytic substrate peak, and appearance time is about 3.7min, b peak and is
Its catalysate peak, appearance time is about 12.8min；
Fig. 5 is FUT8 catalytic reaction ideograph.
Detailed description of the invention
Below in conjunction with accompanying drawing and concrete condition, detailed description of the invention is elaborated.
The present invention, in being embodied as, comprises the following steps:
(1) according to Protein Data Bank (UniProtKB-Q9BYC5) people source core fucosyltransferase structure
Information design fusion protein sequence, to realize utilizing Pichia sp. secreting, expressing core fucosyltransferase.Remove core rock
The trans-membrane region that algae glycosyl transferase N end is combined with Golgi membrane, and at the N end introducing alpha signal peptide of residue sequence
(Native saccharomyces cereisiae secretion signal), introduces 6 His aminoacid for dividing at C end
From purification tag.Sequence table SEQ ID No.1；
Nucleoside according to Pichia sp. codon preference synthesizing recombined human source core fucosyltransferase FUT8 gene
Acid sequence table SEQ ID No.2；
Described Pichia sp. is that (commercially available prod, such as ACCC Chinese agriculture microorganism fungus kind for Pichia yeast cell X-33
Preservation administrative center, ISF China Agriculture Academe Fertilizer Institute or CGMCC General Microbiological Culture preservation administrative center
The bacterial strain of preservation)；
(2) the core fucosyltransferase gene of synthesis and expression matter in digestion with restriction enzyme step (1) are utilized
Grain carrier pPICZ α A, builds recombinant expression plasmid pPICZ α A-FUT8, and method is, by the gene order core fucose of synthesis
Based transferase FUT8 and plasmid vector pPICZ α A utilizes restricted enzyme to carry out enzyme action, reclaims purpose fragment, utilizes T4DNA
The endonuclease bamhi of the endonuclease bamhi of core fucosyltransferase FUT8 with plasmid vector pPICZ α A is connected by ligase, obtains
Recombinant expression plasmid pPICZ α A-FUT8, is transformed in escherichia coli TP10 and expands；
(3) restricted enzyme is utilized to convert Pichia sp. after recombinant expression carrier pPICZ α A-FUT8 carries out linearisation
The competent cell of X-33, the application YPDS flat screen containing antibiotic selects induction type positive transformant；
(4) the fermentation screening of recombination human source core fucosyltransferase FUT8 Pichi strain is expressed: from step (3)
When the induction type positive transformant filtered out is cultivated to OD600=2-6 in BMGY culture medium, BMMY culture medium carries out egg of recombinating
White abduction delivering, induces 2-4 days, and fermented liquid supernatant with SDS-PAGE detection protein expression amount and is examined by Western blot method
Whether be core fucosyltransferase albumen, filter out induction type high-level secretory expression recombination human source core fucose base if surveying
The Pichia sp. recombinant bacterial strain of transferring enzyme FUT8；
(5) isolated and purified recombination human source core fucosyltransferase in Pichia sp. recombinant bacterial strain fermented supernatant fluid
FUT8, the Pichia sp. recombinant bacterial strain of recombination human source core fucosyltransferase FUT8 step (4) filtered out lures
Lead expression 2-4 days, fermented supernatant fluid Ni-NTA SepharoseTMExcel column one-step method is purified, it is thus achieved that purification
Recombination human source core fucosyltransferase FUT8.
The concrete operations of each step are as follows；
Described in step (1) according to Pichia sp. codon preference synthesize core fucosyltransferase FUT8 gene
Nucleotide sequence, be on the basis of not changing core fucosyltransferase FUT8 aminoacid sequence, according to Pichia sp.
Core fucosyltransferase FUT8 complete genome sequence is optimized and synthesizes by Preference codon, simultaneously at core fucose
Based transferase FUT8 5 ' end and 3 ' ends introduce restriction enzyme site, add protein tag sequence side before the termination codon of C end
Protein purification continuous after an action of the bowels, protein tag is histidine (6XHis).
Structure recombinant expression carrier pPICZ α A-FUT8 described in step (2), method is, by synthesis gene order with
Plasmid vector pPICZ α A utilizes restricted enzyme to carry out double digestion, reclaims purpose fragment, utilizes T4DNA ligase by FUT8
Endonuclease bamhi be connected with plasmid pPICZ α A endonuclease bamhi, obtain recombinant expression plasmid pPICZ α A-FUT8, be transformed into large intestine bar
Bacterium TP10 expands；
Described double digestion is to utilize restricted enzyme X hoI and X baI by the core fucose of synthesis in step (1)
Based transferase gene FUT8 and inducible expression vector pPICZ α A carries out double digestion, and gel electrophoresis reclaims target dna sheet after separating
Section, then utilizes T4DNA ligase that DNA fragmentation carries out 16 DEG C and connects overnight, and 42 DEG C of thermal shocks convert escherichia coli TP10 impression
State cell, coating, containing the LB culture medium flat plate of 25ug/ml Zeocin, is placed in overnight incubation at 37 DEG C, random picking monoclonal,
Extracting recombinant expression plasmid pPICZ α A-FUT8, identifies positive colony by double digestion, determines that double digestion post-fragment size is
The no target gene size that meets, whether the clone namely detecting screening contains target plasmid, LB culture medium be by yeast powder 5g,
10g peptone, 5g sodium chloride, regulation pH value, to 7.2-7.4, adds water to 1000ml and makes.
Conversion pichia pastoris X-33 competent cell described in step (3), method is, uses electroporated or chemical turn
Changing, electroporated is to utilize restricted enzyme that recombinant plasmid pPICZ alpha A-FUT8 built is carried out linearisation, uses electrotransfer
Device electroporated Pichi strain competent cell；Chemical Screening is to utilize plate screening positive transformant, containing corresponding anti-
The YPDS plate screening of raw element obtains induction type positive transformant；
Described YPDS flat board is by 10g yeast powder, 20g peptone, 20g-D glucose, 15g agar, 10g sorbitol
Add water to 1000ml make；
Described in step (3) expression plasmid pPICZ α A-FUT8 is carried out linearisation is to utilize restricted enzyme PmeI
Enzyme action inducible expression vector pPICZ α A-FUT8, taking 5-20ug plasmid, to carry out electroporated pichia pastoris X-33 competence thin
Born of the same parents, the parameter of eppendorf-Eporator electricity conversion instrument used is: voltage 1.5KV, electric capacity hinders 200 Ω, discharge time
4.8-5.2ms, adds 1.0M sorbitol ice-cold for 1.0ml immediately after electric shock, be coated with and contain after 30 DEG C of shaken cultivation 1-2h
The YPDS flat board of 100ug/ml Zeocin, grows transformant after being placed in 28-30 DEG C of cultivation 2-4 days.
Screening described in step (4) obtains induction type excreting and expressing recombinant human source core fucosyltransferase FUT8's
Pichia sp. recombinant bacterial strain, the Pichia sp. of described induction type excreting and expressing recombinant human source core fucosyltransferase FUT8
Recombinant bacterial strain is to be inoculated in culture medium, by shake flask fermentation abduction delivering in flat board picking transformant, it is thus achieved that hypersecretion is expressed
The Pichi strain of recombination human source core fucosyltransferase FUT8；
Wherein；Described culture medium includes BMGY/BMMY；
Described BMGY culture medium is: 10g yeast powder, 20g peptone, the 100mM phosphate buffer of pH6.0,13.4g without
Aminoacid yeast nitrogen, 4X10-4Biotin, 10ml glycerol, add water to 1000ml and make；
Described BMMY culture medium is: 10g yeast powder, 20g peptone, the 100mM phosphate buffer of pH6.0,13.4g without
Aminoacid yeast nitrogen, 4X10-4Biotin, 5ml methanol, add water to 1000ml and make；
Isolated and purified recombinant core fucosyltransferase FUT8 from fermentation medium described in step (5) is: will
After the Pichi strain fermentation culture of the high level expression recombiant protein obtained, the centrifuged supernatant of fermentation liquid, according to table
The recombiant protein that reaches select with the Ni-NAT chromatographic column of His label, first protein crude extract is gone up Ni-NAT chromatographic column,
The albumen that not can be incorporated on pillar is washed away, then with the level pad containing different salt ionic concentrations with level pad
Eluting meticulously, collects eluent, the effect of application SDS-PAGE electrophoresis detection protein purification, uses the method for dialysis or use desalination
The mode desalination of post eluting.
With 10 column volumes of equilibration buffer Ni-NAT chromatographic column, by the protein sample after above-mentioned dialysis equilibrium
Post, carries out affine displacement chromatography, and after sample upper prop, continuation level pad washes 20 column volumes, the most respectively with containing
Have 40,60,80,100,120 and the level pad of 200mM imidazole concentration carry out gradient stepwise elution, collect eluent, i.e. obtain
Obtain recombiant protein FUT8 after purification, with the mode desalination of desalting column eluting, detect enzymatic activity.
From the above, it is seen that the present invention includes three parts: recombination human source core fucosyltransferase FUT8 finishes red
The structure of yeast secreted expression system, the isolated and purified and recombination human source core of recombination human source core fucosyltransferase FUT8
The detection of fucosyltransferase FUT8 enzymatic activity, existing according to above-mentioned concrete operation step and Figure of description, the present invention is entered
One step illustrates.
The structure of embodiment 1 recombination human source core fucosyltransferase FUT8 secreting, expressing system
(1) according to Protein Data Bank (UniProtKB-Q9BYC5) people source core fucosyltransferase structure
Information design fusion protein sequence, to realize utilizing Pichia sp. secreting, expressing core fucosyltransferase.Remove core rock
The trans-membrane region that algae glycosyl transferase N end is combined with Golgi membrane, and at the N end introducing alpha signal peptide of residue sequence
(Native saccharomyces cereisiae secretion signal), introduces 6 His aminoacid for dividing at C end
From purification tag.Sequence table SEQ ID No.1；
Nucleoside according to Pichia sp. codon preference synthesizing recombined human source core fucosyltransferase FUT8 gene
Acid sequence table SEQ ID No.2；
By on above Fusion gene construction to yeast expression vector, the recombiant protein of fusion will be at pichia pastoris X-33
Interior expression: the gene above-mentioned design synthesized, is building up on yeast expression vector pPICZ α A with XhoI and Xba I, obtains
Recombinant vector pPICZ α A-FUT8；(synthetic gene is completed by Suzhou Bioisystech Co., Ltd of saving worry)
Embodiment 2 builds induction type recombinant expression plasmid pPICZ α A-FUT8
Described double digestion is to utilize restricted enzyme X hoI and X baI by the core fucose of synthesis in step (1)
Inducible expression vector pPICZ α A after based transferase FUT8 gene and double digestion utilizes T4DNA ligase to carry out DNA fragmentation
16 DEG C connect overnight, and 42 DEG C of thermal shocks convert escherichia coli TP10 competent cell, and the coating LB containing 25ug/ml Zeocin cultivates
Base flat board, is placed in overnight incubation at 37 DEG C, random picking monoclonal, extracts recombinant expression plasmid pPICT α A-FUT8, by double
Enzyme action identifies positive colony, as in figure 2 it is shown, it is the recombinant core fucosyltransferase albumen FUT8 induction type table built
Reach plasmid vector pPICT α A-FUT8 schematic diagram；
Above-mentioned LB culture medium is by yeast powder 5g, 10g peptone, 5g sodium chloride, and regulation pH value, to 7.2-7.4, adds water to
1000ml makes.
Embodiment 3 Pichi strain converts and screening
Utilize restricted enzyme PmeI enzyme action inducible expression vector pPICZ α A-FUT8, take 5-20ug plasmid and carry out electricity
Hitting conversion pichia pastoris X-33 competent cell, the parameter of eppendorf-Eporator electricity conversion instrument used is: voltage
1.5KV, electric capacity hinders 200 Ω, and discharge time, 4.8-5.2ms, added 1.0M sorbitol ice-cold for 1.0ml immediately after electric shock, 30 DEG C
The coating YPDS flat board containing 100ug/ml Zeocin after shaken cultivation 1-2h, grows conversion after being placed in 28-30 DEG C of cultivation 2-4d
Son.
Described YPDS flat board is by 10g yeast powder, 20g peptone, 20g-D glucose, 15g agar, 10g sorbitol
Add water to 1000ml make；
Embodiment 4 high-level secretory expression recombination human source core fucosyltransferase FUT8 Pichia sp. recombinant bacterial strain
Screening
The induction type positive transformant filtered out from step (3) on YPDS flat board streak culture 2-3 days, picking list bacterium
Falling to being inoculated in the 5ml centrifuge tube containing 1ml BMGY, 30 DEG C of 200rpm cultivate 2-4 days, every in BMMY culture medium culturing bacterial strain
24h adds methanol final concentration of 0.5%, and cultivates at 30 DEG C of 200rpm.After induction 2-4d, 5000rpm is centrifugal collects supernatant.With
Whether SDS-PAGE detection expressing quantity the albumen expressed with the detection of Western blot method are core fucosyltransferase
Figure (3), each sample parallel two row detects, and final screening obtains high level the Pichia sp. of correct secreting, expressing FUT8
Bacterial strain.
Described BMGY culture medium is: 10g yeast powder, 20g peptone, 10g sorbitol, the 100mM phosphoric acid buffer of pH6.0
Liquid, 13.4g are without aminoacid yeast nitrogen, 4X10-4Biotin, add water to 1000ml and make；
Described BMMY culture medium is: 10g yeast powder, 20g peptone, the 100mM phosphate buffer of pH6.0,13.4g without
Aminoacid yeast nitrogen, 4X10-4Biotin, 5ml methanol, add water to 1000ml and make.
Screened by above-mentioned fermentation, it is thus achieved that there is hypersecretion and express finishing of recombination human source core fucosyltransferase FUT8
Red yeast strain pPICZ α A-FUT8/X-33.Through inspection, as shown in Figure 3 B, from Pichia sp., successfully obtain recombination human source
Core fucosyltransferase FUT8.
Embodiment 5 recombinant core fucosyltransferase isolated and purified
The hypersecretion that has step (4) screening obtained expresses the complete red of recombination human source core fucosyltransferase FUT8
Yeast recombinant strain strain carries out fermentation inducement and expresses 2-4 days, 5000rpm, 4 DEG C fermentation liquid is centrifuged 10min, collect supernatant,
Use Ni-NTA SepharoseTMExcel column one-step method is purified.
With 10 column volumes of equilibration buffer Ni-NAT chromatographic column, by the protein sample after above-mentioned dialysis equilibrium
Post, carries out affine displacement chromatography, and after sample upper prop, continuation level pad washes 20 column volumes, the most respectively with containing
Have 40,60,80,100,120 and the level pad of 200mM imidazole concentration carry out gradient stepwise elution, collect eluent, i.e. obtain
Obtaining recombiant protein FUT8 after purification, the gene structure display of recombiant protein FUT8 as shown in Figure 3A, detects with SDS-PAGE
The effect of protein purification；
Core fucosyltransferase after purification uses the method desalination of desalting column, cryopreservation.
Embodiment 6 recombination human source core fucosyltransferase FUT8 Enzyme assay
The activity of the recombination human source core fucosyltransferase FUT8 prepared by inspection above-mentioned steps (5), uses former
Reason and method, process is as follows:
This experiment is to utilize core seven sugar structure GnGn-bi-Asn-PABA with fluorophor as recombination human source core
Heart fucosyltransferase FUT8 catalytic substrate, in the reaction system containing recombination human source core fucosyltransferase FUT8
In, through the product with the catalytic reaction generation of substrate again with fluorescent labeling, utilize HPLC pair with fluorescence detector
Catalytic substrate separates with product, detection.As shown in Figure 4, the recombination human source core fucosyltransferase FUT8 of expression has sky
So biologic activity, modifies for the external oligosaccharide of core fucosyltransferase FUT8 and lays a good foundation.
HPLC methods analyst: take the core fucosyltransferase albumen after 5ul desalination and carry out HPLC detection.Chromatograph institute
Pump be Waters 1525Binary HPLC Pump, detector is Waters 474Scanning Fluorescence
Detectoer, chromatographic column is Nova-Pack C18 3.9X150mm (Waters), column temperature 55 DEG C.Exciting light/detection light wave
A length of 320/400nm, eluent flow rate is 1ml/min, tests complete water: methanol (80:20) rinses chromatographic column 40min.Profit
With Breeze software, chromatogram image is analyzed, according to the relative catalytic activity of product peak-to-peak areal calculation unit concentration FUT8
Figure (4).Reaction system is 10ul:10uM GnGn-bi-Asn-PABA, 0.5mM GDP-L-fucose, 200Mm MES-NaOH
PH 7.0,5ul core fucosyltransferase.
From the above, it is seen that the present invention has following significant advantage: the recombiant protein of expression eliminates at Golgi body
Retention signal on film makes core fucosyltransferase successful secretion in culture medium；To gene order FUT8 according to complete red ferment
The Preference of female codon is optimized so that it is be more suitable in Pichia sp. expressing；The recombiant protein expressed is with special
Label, it is easy to carry out the isolated and purified of recombiant protein；After isolated and purified in Pichia sp. recombinant bacterial strain expression supernatant
FUT8 has enzymatic activity, is used directly for external oligosaccharide and modifies.
Sequence table
<110>University Of Dalian
<120>method that pichia yeast expression system secretion produces people source core fucosyltransferase is utilized
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 564
<212> PRT
<213>synthetic
<400> 1
RFPSIFTAV LFAASSALAA PVNTTTEDET AQIPAEAVIG YSDLEGDFDV AVLPFSNSTN 60
NGLLFINTTI ASIAAKEEGV SLEKREAEAG LGKDHEILRR RIENGAKELW FFLQSELKKL 120
KNLEGNELQR HADEFLLDLG HHERSIMTDL YYLSQTDGAG DWREKEAKDL TELVQRRITY 180
LQNPKDCSKA KKLVCNINKG CGYGCQLHHV VYCFMIAYGT QRTLILESQN WRYATGGWET 240
VFRPVSETCT DRSGISTGHW SGEVKDKNVQ VVELPIVDSL HPRPPYLPLA VPEDLADRLV 300
RVHGDPAVWW VSQFVKYLIR PQPWLEKEIE EATKKLGFKH PVIGVHVRRT DKVGTEAAFH 360
PIEEYMVHVE EHFQLLARRM QVDKKRVYLA TDDPSLLKEA KTKYPNYEFI SDNSISWSAG 420
LHNRYTENSL RGVILDIHFL SQADFLVCTF SSQVCRVAYE IMQTLHPDAS ANFHSLDDIY 480
YFGGQNAHNQ IAIYAHQPRT ADEIPMEPGD IIGVAGNHWD GYSKGVNRKL GRTGLYPSYK 540
VREKIETVKY PTYPEAEKHH HHHH 564
<210> 2
<211> 1452
<212> DNA
<213>synthetic
<400> 2
ttgggtaagg atcacgagat cctgagacgt agaatcgaga acggagcaaa agaattgtgg 60
ttcttcctgc aatccgagct gaagaagctg aagaacctgg aaggtaacga gttgcaaaga 120
cacgctgacg agttcttgct ggatttgggt catcacgagc gttctatcat gaccgacctg 180
tactacctgt ctcaaactga cggagctgga gattggagag aaaaagaagc taaggacttg 240
accgagttgg ttcagcgtag aatcacctac ctgcagaacc caaaggactg ttctaaggcc 300
aagaagctgg tctgcaacat caacaagggt tgcggatacg gttgccaatt gcatcacgtc 360
gtctactgtt tcatgatcgc ctacggtacc cagagaactc tgatcttgga gtcccagaac 420
tggagatacg ctactggagg ttgggaaacc gttttccgtc cagtttctga gacctgtacc 480
gatagatccg gtatctccac tggtcattgg tctggagaag tcaaggacaa gaacgtccag 540
gttgttgagc tgccaatcgt tgactctttg cacccaagac caccatactt gccattggct 600
gttccagaag acttggcaga cagattggtc agagttcacg gagatcctgc tgtttggtgg 660
gtctctcaat tcgtcaagta cctgatccgt ccacaacctt ggttggaaaa agaaattgaa 720
gaagctacta agaagctggg tttcaagcac cctgtcatcg gagttcacgt tagaagaacc 780
gacaaggtcg gtactgaagc tgctttccat ccaatcgaag aatacatggt ccacgttgaa 840
gagcacttcc aattgcttgc ccgtagaatg caggtcgata agaagagagt ctacctggct 900
accgacgatc catctttgtt gaaagaagct aagaccaagt acccaaacta cgagttcatc 960
tccgacaact ccatctcttg gtctgctggt ttgcacaacc gttacactga gaactccctg 1020
agaggtgtta tcctggacat ccacttcttg tcccaagctg acttcttggt ctgcactttc 1080
tcctctcagg tctgtagagt tgcctacgag atcatgcaga ccttgcatcc agacgcttct 1140
gctaacttcc actctctgga cgacatctac tacttcggag gtcaaaacgc tcacaaccag 1200
atcgctatct acgctcatca gccaagaact gctgacgaaa tcccaatgga gccaggagac 1260
atcattggag ttgctggaaa ccattgggac ggttattcca agggagtcaa ccgtaagttg 1320
ggtagaaccg gattgtaccc atcctacaag gtcagagaga agatcgagac cgtcaagtac 1380
ccaacctacc cagaagctga aaaggctgga ggtggtcatc atcaccacca cggtggtggt 1440
taatagtcta ga 1452
Claims (8)
1. utilize the method that pichia yeast expression system secretion produces people source core fucosyltransferase, it is characterised in that bag
Include following steps:
Step 1: believe according to Protein Data Bank (UniProtKB-Q9BYC5) people source core fucosyltransferase structure
Breath design fusion protein sequence, utilizes RCSB PDB to announce core fucosyltransferase Protein Information and removes core fucose base
The trans-membrane region that transferring enzyme N end is combined with Golgi membrane, and at the N end introducing alpha signal peptide of residue sequence, at C end
Introduce 6 His aminoacid before termination codon, for isolated and purified label, facilitate follow-up protein purification, protein tag
For histidine (6XHis), amino sequence table SEQ ID No.1；
Utilize Pichia sp. codon preference to synthesize core fucosyltransferase FUT8 gene, be not change core rock algae
On the basis of glycosyl transferase FUT8 aminoacid sequence, according to Pichia sp. Preference codon to core fucosyltransferase
FUT8 complete genome sequence is optimized and synthesizes, and simultaneously at the 5 of core fucosyltransferase FUT8, end and 3, end introduces enzyme action
Site, its nucleotides sequence list is SEQ ID No.2；
Described Pichia sp. is Pichia yeast cell X-33；
Step 2: utilizing restricted enzyme to build recombinant expression plasmid pPICZ α A-FUT8, method is: by synthesis in step (1)
Recombination human source core fucosyltransferase FUT8 and expression plasmid carrier pPICZ α A, utilize restricted enzyme to carry out enzyme
Cut, reclaim purpose fragment, utilize T4 DNA ligase to be connected with plasmid vector X endonuclease bamhi by the endonuclease bamhi of FUT8, obtain
Recombinant expression plasmid pPICZ α A-FUT8, is transformed in escherichia coli TP10 and expands；
Step 3: utilize restricted enzyme to convert Pichia sp. after recombinant expression carrier pPICZ α A-FUT8 carries out linearisation
The competent cell of bacterium cell X-33, the application YPDS flat screen containing antibiotic selects induction type positive transformant；
Step 4: express the fermentation screening of recombiant protein FUT8 Pichi strain: the induction type positive that step (3) filters out turns
When beggar cultivates OD600=2-6 in BMGY culture medium, changing culture medium is the abduction delivering that BMMY carries out recombiant protein, induction
2-4 days, fermented liquid supernatant with SDS-PAGE detection protein expression amount and detected whether as core rock algae by Western blot method
Glycosyl transferase, filters out the Pichia sp. of induction type high-level secretory expression recombination human source core fucosyltransferase FUT8
Recombinant bacterial strain；
Step 5: isolated and purified recombination human source core fucosyltransferase FUT8 in Pichia sp. recombinant bacterial strain fermented supernatant fluid.
The most according to claim 1 pichia yeast expression system secretion is utilized to produce people source core fucosyltransferase
Method, it is characterised in that the concrete operation step of step 2 is: utilize restricted enzyme X hoI and X baI by step (1)
The core fucosyltransferase gene FUT8 and inducible expression vector pPICZ α A of synthesis carry out double digestion, and gel electrophoresis divides
PPICZ α A linear fragment gene after the rear i.e. double digestion of recovery target DNA fragments, then utilizes T4 DNA ligase to target
DNA fragmentation carries out 16 DEG C and connects overnight, and 42 DEG C of thermal shocks convert escherichia coli TP10 competent cell, and coating is containing 25ug/ml
The LB culture medium flat plate of Zeocin, is placed in overnight incubation at 37 DEG C, random picking monoclonal, extracts recombinant expression plasmid pPICZ α
A-FUT8, identifies positive colony by double digestion.
The most according to claim 2 pichia yeast expression system secretion is utilized to produce people source core fucosyltransferase
Method, it is characterised in that described LB culture medium is by yeast powder 5g, 10g peptone, 5g sodium chloride, regulation pH value to 7.2-
7.4, add water to 1000ml and make.
The most according to claim 1 pichia yeast expression system secretion is utilized to produce people source core fucosyltransferase
Method, it is characterised in that the concrete operations of step 3 are: utilize restricted enzyme PmeI enzyme action inducible expression vector pPICZ
α A-FUT8, takes 5-20ug plasmid and carries out electroporated pichia pastoris X-33 competent cell, eppendorf-used
The parameter of Eporator electricity conversion instrument is: voltage 1.5KV, and electric capacity hinders 200 Ω, and discharge time, 4.8-5.2ms, added after electric shock immediately
Enter 1.0M sorbitol ice-cold for 1.0ml, the coating YPDS flat board containing 100ug/ml Zeocin after 30 DEG C of shaken cultivation 1-2h,
Induction type positive transformant is grown after being placed in 28-30 DEG C of cultivation 2-4d.
The most according to claim 4 pichia yeast expression system secretion is utilized to produce people source core fucosyltransferase
Method, it is characterised in that described YPDS flat board is by 10g yeast powder, 20g peptone, 20g-D glucose, 15g agar,
10g sorbitol adds water to 1000ml and makes.
The most according to claim 1 pichia yeast expression system secretion is utilized to produce people source core fucosyltransferase
Method, it is characterised in that the concrete operations of step 4 are: the induction type positive transformant filtered out from step 3 is at YPDS flat board
Upper streak culture 2-3 days, picking list colony inoculation was in the 5ml centrifuge tube containing 1ml BMGY culture medium, and 30 DEG C of 200rpm train
When supporting OD600=2-6, changing culture medium is the abduction delivering that BMMY carries out recombiant protein, every 24h in BMMY culture medium culturing bacterial strain
Adding methanol final concentration of 0.5%, and cultivate at 30 DEG C of 200rpm, after induction 2-4d, 5000rpm is centrifugal collects supernatant；Use SDS-
PAGE detection expressing quantity also detects by Western blot method, and each sample parallel two row detects, and finally screens
Obtain high level the Pichi strain of correct excreting and expressing recombinant human source core fucosyltransferase FUT8.
The most according to claim 6 pichia yeast expression system secretion is utilized to produce people source core fucosyltransferase
Method, it is characterised in that described BMGY culture medium is: 10g yeast powder, 20g peptone, 10g sorbitol, the 100mM of pH6.0
Phosphate buffer, 13.4g are without aminoacid yeast nitrogen, 4X10-4Biotin, add water to 1000ml and make；
Described BMMY culture medium is: 10g yeast powder, 20g peptone, the 100mM phosphate buffer of pH6.0,13.4g are without amino
Acid leaven nitrogen source, 4X10-4Biotin, 5ml methanol, add water to 1000ml and make.
The most according to claim 1 pichia yeast expression system secretion is utilized to produce people source core fucosyltransferase
Method, it is characterised in that the concrete operations of step 5 are: the hypersecretion that has that step 4 is screened acquisition expresses recombination human source core
The Pichia sp. recombinant bacterial strain of fucosyltransferase FUT8 carries out fermentation inducement expresses 2-4 days, and at 5000rpm, 4 DEG C to fermentation
Liquid is centrifuged 10min, collects supernatant, uses Ni-NTA SepharoseTMExcel column one-step method is purified, it is thus achieved that pure
The recombiant protein FUT changed；
With 10 column volumes of equilibration buffer Ni-NAT chromatographic column, by the protein sample upper prop after above-mentioned dialysis equilibrium, enter
The affine displacement chromatography of row, after sample upper prop, continuation level pad washes 20 column volumes, the most respectively with containing 40,
60,80,100,120 and the level pad of 200mM imidazole concentration carry out gradient stepwise elution, collect eluent, i.e. obtain pure
Recombiant protein FUT8 after change, by the effect of SDS-PAGE detection protein purification；
Core fucosyltransferase after purification uses the method desalination of desalting column, cryopreservation.
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| CN106399360A (en) * | 2015-07-27 | 2017-02-15 | 上海药明生物技术有限公司 | FUT8 gene knockout method based on CRISPR technology |
| CN108048510A (en) * | 2018-02-02 | 2018-05-18 | 广东海纳川生物科技股份有限公司 | A kind of purifying process of thanatin antibacterial peptide |
| CN108251437A (en) * | 2017-11-30 | 2018-07-06 | 中山珐玛斯医药科技有限公司 | A kind of method for improving cyp3A5 expression enzyme metabolic activities |
| CN116769746A (en) * | 2023-05-12 | 2023-09-19 | 康彤(上海)生物研发有限公司 | Method for improving ability of pichia pastoris to secrete cyclodextrin glucosyltransferase |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106399360A (en) * | 2015-07-27 | 2017-02-15 | 上海药明生物技术有限公司 | FUT8 gene knockout method based on CRISPR technology |
| CN108251437A (en) * | 2017-11-30 | 2018-07-06 | 中山珐玛斯医药科技有限公司 | A kind of method for improving cyp3A5 expression enzyme metabolic activities |
| CN108048510A (en) * | 2018-02-02 | 2018-05-18 | 广东海纳川生物科技股份有限公司 | A kind of purifying process of thanatin antibacterial peptide |
| CN116769746A (en) * | 2023-05-12 | 2023-09-19 | 康彤(上海)生物研发有限公司 | Method for improving ability of pichia pastoris to secrete cyclodextrin glucosyltransferase |
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