CN110438022A - A kind of precursor protein Kex2 is overexpressed Pichi strain and its construction method - Google Patents
A kind of precursor protein Kex2 is overexpressed Pichi strain and its construction method Download PDFInfo
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Abstract
The present invention relates to a kind of precursor protein Kex2 to be overexpressed Pichi strain and its construction method.It is characterized in that design PCR upstream primer and downstream primer, are carrying out Kex2 gene cloning, are usingEcoR I restriction enzyme respectively to after recovery purifying Kex2 gene and pGAPZB expression plasmid carry out digestion, purify the pGAPZB expression plasmid of the Kex2 gene after digestion and linearisation after digestion respectively with DNA glue purification QIAquick Gel Extraction Kit, then Kex2 gene is connected on pGAPZB expression plasmid using T4 ligase and obtains transformant, transformant connection product is converted and obtains Kex2 overexpression Pichi strain in Escherichia coli TOP 10；The Kex2 for the precursor protein that the present invention constructs is overexpressed Pichi strain, precursor protein can be more effectively processed compared with the traditional Pichia pastoris not being transformed, processing efficiency can reach 85% or more.
Description
Technical field
The invention belongs to gene engineering technology fields, are overexpressed Pichi strain more particularly, to Kex2, especially
Refer to that one kind can highly-efficient processing mammalian precursor albumen Kex2 overexpression Pichi strain and its construction method.
Background technique
Many secreted proteins are all to translate into the biggish precursor protein of molecular weight first in mammal, are then being secreted
By precursor protein invertase, for shearing at the maturation protein for having physiological activity, furin is it on specific site in channel
The critically important precursor protein invertase of middle one kind.Precursor protein is made of signal peptide, leader peptide and mature peptide.Before signal peptide guidance
Body protein enters endoplasmic reticulum cavity, and leader peptide is most important to the folding of mature peptide and activity.Between leader peptide and mature peptide
There is a conservative precursor protein invertase restriction enzyme site, for precursor protein in maturation, it is logical that leader peptide can be located in secretion
Precursor protein enzyme on road removes, and finally secrets out of the maturation protein with physiological function.
Comprising largely having the active albumen of important Physiological effect and polypeptide in the secretory precursor protein of furin processing, such as
Peptides hormone, nerve polypeptide, insulin and its receptor, Growth and Differentiation Factors, cell surface receptor, extracellular metalloprotein
Enzyme, coagulation factor, adhesion molecule, plasma protein etc., wherein a large portion is pharmaceutical protein candidate or has been successful
Protein drug.
Content is humble in vivo for these secreted proteins, largely can not isolate and purify acquisition by natural material.
To obtain this large amount of albuminoid for research or clinical application, ideal solution is that foreign protein system is utilized to carry out
Recombinant expression.Prokaryotes do not have endoplasmic reticulum is such to secrete processing channel, do not have the ability of processing precursor protein.Lactation is dynamic
Object cellular expression levels are low, and culture is complicated, at high cost, provide no advantage against in production.Pichia pastoris has both prokaryotes and true
The advantage of core biology all has very big advantage when expressing foreign protein in cost and yield.However Pichia pastoris does not have
Furin gene, processing, this albuminoid timeliness rate is low.Therefore develop it is a kind of can highly-efficient processing mammalian precursor albumen finish
Red yeast strain produces this kind of secretory protein and application is of great significance.
Summary of the invention
The purpose of the present invention is for above existing problem and shortage, providing one kind can highly-efficient processing mammalian precursor egg
White Kex2 is overexpressed Pichi strain and its construction method.This method clones Kex2 gene from Pichia pastoris genome；
Kex2 gene is connect with yeast expression vector, constructs gene recombination plasmid containing Kex2；By the recombinant plasmid digestion of building
After linearisation, import in Pichia pastoris；Kex2 gene integration is determined into Pichia pastoris genome and is expressed, has been acquired
The Kex2 of function is overexpressed Pichi strain.It, can be effectively by the leader peptide in precursor protein from mature peptide by the above method
In cut off, final successful expression secrets out of maturation protein.
The present invention is achieved by the following scheme:
An object of the present invention be to provide it is a kind of can the Kex2 of highly-efficient processing mammalian precursor albumen be overexpressed Pichia pastoris
Bacterial strain.The method used to achieve the purpose of the present invention be by Pichia pastoris Kex2 gene, DNA sequence dna as described in SEQ1,
The protein sequence of coding is imported into Pichia pastoris after connecting with the expression vector of Pichia pastoris as shown in SEQ2, be built into and
Kex2 is overexpressed Pichi strain.The bacterial strain expressing K ex2 precursor protein invertase, can highly-efficient processing mammalian precursor egg
It is white.
The second object of the present invention is to provide a kind of construction method of precursor protein Kex2 overexpression Pichi strain.For
Achieve the object of the present invention the technical solution adopted is as follows:
1, PCR primer designs:
1) upstream primer:
Kex2-up:5'-GGGGAATTCAtgTATTTGCCAGCACTTCGCTTA-3', dashed part are EcoR I restriction enzyme
Enzyme site；
2) downstream primer
Kex2-down:5'-GGGGAATTCTtacaat gccgcacgtttgggatg-3', dashed part are EcoR I restriction enzyme
Restriction enzyme site.
2, Kex2 gene cloning
1) in superclean bench, one Pichia pastoris GS115 single bacterium of inoculation falls on the conical flask containing 5mLYPD fluid nutrient medium
In, 30 DEG C of culture 24-36h obtain Pichia pastoris culture solution.
2) take 1ml culture to OD600=1.0~2.0 Pichia pastoris culture solution, 1500g, 4 DEG C of centrifugation 5min collect bacterium
Body.
The DNA extraction kit is purchased from TaKaRa company.
3) the Pichia pastoris GS115 gene DNA for drawing 100ng-500ng is template, carries out PCR amplification and obtains target gene
Product.
The PCR amplification condition are as follows: 94 DEG C of 5min of initial denaturation；It is denaturalized 94 DEG C of 30sec；Anneal 55 DEG C of 30sec；Extend 72
℃3min；30 circulations；72 DEG C of extension 5min.
4) recovery purifying is carried out to target gene product using DNA glue purification QIAquick Gel Extraction Kit, obtains Kex2 after purification
Gene.
The DNA glue purification QIAquick Gel Extraction Kit is bought in promega company.
5, Kex2 is overexpressed the preparation of Pichi strain
With EcoR I restriction enzyme respectively to after recovery purifying Kex2 gene and pGAPZB expression plasmid carry out digestion, digestion is complete
Afterwards, the pGAPZB expression plasmid of the Kex2 gene after digestion and linearisation is purified respectively with DNA glue purification QIAquick Gel Extraction Kit, then
Kex2 gene is connected on pGAPZB expression plasmid using T4 ligase, transformant is obtained, transformant connection product is converted
Kex2 is obtained in Escherichia coli TOP 10 is overexpressed Pichi strain.
The transformant send Sangon Biotech (Shanghai) Co., Ltd. to carry out sequencing analysis, sequence and connection side
It is named as pGAPZB-Kex2 to correct carrier, the building of Pichi strain is overexpressed for Kex2.
Specific pastoris genomic dna extraction, gene downlink connection and step of converting reference product specification in above-mentioned steps
It carries out.
The T4 ligase is purchased from TaKaRa company.
Genomic DNA is extracted in above-mentioned technical proposal, recovery purifying by specification operating procedure carries out.
Transformant of the present invention, preparation process are as follows:
1) plasmid linearization, process are: it is linear to carry out digestion to recombinant plasmid pGAPZB-Kex2 using restriction enzyme Avr II
Change, the plasmid pGAPZB-Kex2 after purification and recovery linearisation is carried out using DNA glue purification QIAquick Gel Extraction Kit, in order to guarantee to convert
Efficiency, linearization plasmid after purification should be greater than 100ng/ul or more.- 20 DEG C of the linearization plasmid prepared saves backup.
2) Pichia pastoris GS115 feels state cell, and preparation process is: in superclean bench, being inoculated with a Pichia pastoris
GS115 single bacterium is fallen in the 25mL conical flask containing 5mLYPD fluid nutrient medium, 30 DEG C of 12~16h of culture.Finishing for culture is red
According in the ratio switching big conical flask of the 250mL containing 50mLYPD fluid nutrient medium of 1:1000,30 DEG C are cultivated yeast GS115
12~16h, until OD600=1.3~1.5.1500g, 4 DEG C of centrifugation 5min, collection thallus, sterile pair of thallus 40mL ice bath
Water is steamed to be resuspended.1500g, 4 DEG C of centrifugation 5min, collect thallus, and the aseptic double-distilled water of thallus 25mL ice bath is resuspended.1500g, 4 DEG C
It is centrifuged 5min, collects thallus, the 1M sorbitol solution of thallus 25mL ice bath is resuspended.1500g, 4 DEG C of centrifugation 5min collect bacterium
Body, the 1M sorbitol solution of thallus 0.5mL ice bath are resuspended, Pichia pastoris competent cell.
3) electrotransformation: the linearization plasmid pGAPZB-Kex2 by 5~10 μ g by ice pre-cooling is added to the 80 above-mentioned processing of μ l
In good competent cell, mixing, the plasmid volume of addition does not exceed 20 μ l.It is entire processed in order to improve transformation efficiency
Journey should be completed on ice bath.Mixed liquor is transferred in the conversion cup of preparatory ice bath (0.2cm type), stands 5min on ice.It will be electric
Conversion cup is put into II electric converter of Gene pulser (Bio-Rad company), is carried out electricity by saccharomyces cerevisiae electricity carryover sequence and is turned.Electricity
The cold 1M sorbitol solution of 1mL ice bath is added after turning into conversion cup immediately, in superclean bench, movement gently will conversion
Yeast in cup is resuspended.Conversion fluid is transferred in a new 1.5ml centrifuge tube, 30 DEG C stationary culture 1 hour.Draw culture
200 μ l of conversion fluid afterwards is coated in the plate of YPD containing Zeocin, 30 DEG C of culture carton upside downs, until transformant occurs.
Picking transformant is inoculated into the 25mL conical flask containing 5mLYPD fluid nutrient medium, 30 DEG C of 24~48h of culture.
Pichia pastoris culture solution of the 1ml culture to OD600=2.0~2.5 is taken, 1500g, 4 DEG C of centrifugation 5min collect thallus.Utilize ferment
Female RNA extracts reagent agent box (TaKaRa company) by specification operating procedure and extracts total serum IgE.
The Kex2 that the present invention successfully constructs highly-efficient processing mammalian precursor albumen is overexpressed Pichi strain, real
Verify it is bright, compared with the traditional Pichia pastoris not being transformed, the present invention constructed by Kex2 be overexpressed Pichi strain can more have
Effect processing precursor protein, processing efficiency can reach 85% or more, and traditional Pichia pastoris processing efficiency is less than 30%.Engineering bacteria
Construct successfully for furin processing precursor protein in Pichia pastoris high efficient expression lay a good foundation.
Kex2 gene is as shown in GenBank accession number Accession No CP014716, the gene order such as SEQ1 institute
It states, designs pair of primers according to its coded sequence
Detailed description of the invention
The PCR amplification result electrophoretogram of Fig. 1 Kex2 gene, wherein swimming lane M is DNA molecular amount standard, and swimming lane 1 is Kex2 gene
Pcr amplification product.
Fig. 2 Kex2 gene is expressed in Pichia pastoris, and wherein swimming lane M is DNA molecular amount standard, and swimming lane 1 is building
Kex2 is overexpressed Pichi strain GS115-Kex2；Swimming lane 2 is not turn Kex2 gene Pichia pastoris GS115 bacterial strain.
Fig. 3 Kex2 is overexpressed Pichi strain GS115-Kex2 functional verification report carrier schematic diagram.
Fig. 4 Pichia pastoris GS115 and Kex2 are overexpressed Pichi strain GS115-Kex2 to three furin Processing positions
The precursor protein of (RXXR, RXRR and RXKR, X indicate any amino acid) processes situation, and figure a is Pichi strain
GS115 processes situation to the precursor protein of three furin Processing positions；Scheming b is that recombination Kex2 is overexpressed Pichi strain
GS115-Kex2 processes situation to the precursor protein of three furin Processing positions.
Fig. 5 is sequence table of the present invention.
Specific embodiment
In order to which the objects, technical solutions and advantages of invention are more clearly understood, with reference to the accompanying drawings and embodiments, to the present invention
It is further elaborated.It should be appreciated that specific embodiment is merely to illustrate and explain the present invention, rather than for limiting this
Invention.
Test method without specific conditions in following embodiment is conventional method.
Following bacterial strain and plasmid have been used in the embodiment of the present invention:
Escherichia coli TOP 10 (E.coli TOP 10): for gene cloning operation, purchase is in the silent winged generation that (Thermo of match
Fisher Scientific) company.
Pichia pastoris GS115: purchase is in silent winged generation that (the Thermo Fisher Scientific) company of match.
PGAPZB expression plasmid: it is used for the expressing K ex2 in Pichia pastoris, purchase to be in the silent winged generation that (Thermo of match
Fisher Scientific) company.
The building of embodiment 1Kex2 gene cloning and Pichia pastoris recombinant expression carrier
Kex2 gene as shown in GenBank accession number Accession No CP014716, the gene order as described in SEQ1, according to
Pair of primers PCR primer is designed according to its coded sequence:
Upstream primer
Kex2-up:5'-GGGGAATTCAtgTATTTGCCAGCACTTCGCTTA-3', dashed part are EcoR I restriction enzyme
Enzyme site；
Downstream primer
Kex2-down:5'-GGGGAATTCTtacaat gccgcacgtttgggatg-3', dashed part are EcoR I restriction enzyme
Restriction enzyme site.
In superclean bench, one Pichia pastoris GS115 single bacterium of inoculation falls on the 25mL containing 5mLYPD fluid nutrient medium
In conical flask, 30 DEG C of culture 24-36h.Take 1ml culture to OD600=1.0~2.0 Pichia pastoris culture solution, 1500g, 4 DEG C from
Heart 5min collects thallus.Base is extracted using pastoris genomic dna extracts kit (TaKaRa company) by specification operating procedure
Because of a group DNA.The Pichia pastoris GS115 gene DNA for drawing 100ng-500ng is template, carries out PCR reaction.PCR amplification condition:
94 DEG C of 5min of initial denaturation；It is denaturalized 94 DEG C of 30sec；Anneal 55 DEG C of 30sec；Extend 72 DEG C of 3min；30 circulations；72 DEG C of extensions
5min.1% agarose gel electrophoresis verifies Kex2 gene magnification result (attached drawing 1).
Target gene product is recycled using DNA glue purification QIAquick Gel Extraction Kit (purchase is in promega company), specifically
Operating method refers to kit operation manual.
With EcoR I restriction enzyme respectively to after recovery purifying Kex2 gene and pGAPZB expression plasmid carry out digestion, digestion
After completely, the pGAPZB expression plasmid of the Kex2 gene after digestion and linearisation is purified respectively with DNA glue purification QIAquick Gel Extraction Kit,
Then Kex2 gene is connected on pGAPZB expression plasmid using T4 ligase (TaKaRa company), connection product converts large intestine
Bacillus TOP 10, specific connection and step of converting reference book.The transformant grown on plate send raw work bioengineering (on
Sea) limited liability company's progress sequencing analysis, sequence and the connection correct carrier in direction are named as pGAPZB-Kex2, are used for
The building of Kex2 overexpression Pichi strain.
The building of embodiment 2Kex2 overexpression Pichi strain GS115-Kex2
Plasmid linearization: linearization for enzyme restriction is carried out to recombinant plasmid pGAPZB-Kex2 using restriction enzyme Avr II, utilizes DNA glue
Purification and recovery kit carries out the plasmid pGAPZB-Kex2 after purification and recovery linearisation, in order to guarantee transformation efficiency, after purification
Linearization plasmid should be greater than 100ng/ul or more.- 20 DEG C of the linearization plasmid prepared saves backup.
Pichia pastoris GS115 feels the preparation of state cell: in superclean bench, being inoculated with a Pichia pastoris GS115 single colonie
Into the 25mL conical flask containing 5mLYPD fluid nutrient medium, 30 DEG C of culture 12-16h.By the Pichia pastoris GS115 of culture according to
In the ratio switching big conical flask of the 250mL containing 50mLYPD fluid nutrient medium of 1:1000,30 DEG C of culture 12-16h, until
OD600=1.3~1.5.1500g, 4 DEG C of centrifugation 5min, collect thallus, and the aseptic double-distilled water of thallus 40mL ice bath is resuspended.
1500g, 4 DEG C of centrifugation 5min, collect thallus, and the aseptic double-distilled water of thallus 25mL ice bath is resuspended.1500g, 4 DEG C of centrifugation 5min,
Thallus is collected, the 1M sorbitol solution of thallus 25mL ice bath is resuspended.1500g, 4 DEG C of centrifugation 5min, collect thallus, and thallus is used
The 1M sorbitol solution of 0.5 mL ice bath is resuspended, Pichia pastoris competent cell.
Electrotransformation: the linearization plasmid pGAPZB-Kex2 by 5~10 μ g by ice pre-cooling is added to that 80 μ l are above-mentioned to be handled well
Competent cell in, mixing, the plasmid volume of addition do not exceed 20 μ l.In order to improve transformation efficiency, entire treatment process
It should be completed on ice bath.Mixed liquor is transferred in the conversion cup of preparatory ice bath (0.2cm type), stands 5min on ice.Electricity is turned
Change cup to be put into II electric converter of Gene pulser (Bio-Rad company), carries out electricity by saccharomyces cerevisiae electricity carryover sequence and turn.Electricity turns
The cold 1M sorbitol solution of 1mL ice bath is added into conversion cup immediately afterwards, in superclean bench, movement will gently convert cup
In yeast be resuspended,.Conversion fluid is transferred in a new 1.5ml centrifuge tube, 30 DEG C stationary culture 1 hour.Draw culture
200 μ l of conversion fluid afterwards is coated in the plate of YPD containing Zeocin, 30 DEG C of culture carton upside downs, until transformant occurs.
Embodiment 3 is overexpressed the Pichi strain GS115-Kex2 screening of Kex2 gene
Transformant on picking Zeocin YPD plate as described in example 2, is inoculated into containing 5mLYPD fluid nutrient medium
In 25mL conical flask, 30 DEG C of culture 24-48h.Take 1ml culture to OD600=2.0~2.5 Pichia pastoris culture solution, 1500g,
4 DEG C of centrifugation 5min collect thallus.Reagent agent box (TaKaRa company) by specification operating procedure is extracted using yeast rna to extract
Total serum IgE.(TaKaRa company) by specification operating procedure is taken to synthesize using cDNA synthetic agent box single-stranded needed for RT-PCR
CDNA template.After obtaining single cDNA template, RT-PCR reaction is carried out.1 μ l of cDNA template, reactant in the anti-system of RT-PCR
Other reagent dosage by specifications carry out in system.What the upstream and downstream of RT-PCR was drawn are as follows:
Upstream primer: 5'-ACAGTTCTGGATCGGGTGAG-3'
Downstream primer: 5'-GCCTCCTGGGTCAATTCATA-3'
PCR reaction condition are as follows: 94 DEG C of 5min of initial denaturation；It is denaturalized 94 DEG C of 10sec；Anneal 55 DEG C of 30sec；Extend 72 DEG C of 30 sec；
30 circulations；72 DEG C of extension 5min.1.5% agarose gel electrophoresis verifies amplification, selects and is overexpressed finishing for Kex2 gene
Red yeast strain GS115-Kex2 (attached drawing 2).Kex2 be overexpressed Pichi strain GS115-Kex2 can -80 DEG C of preservations be contained in
In the YPD culture medium of 15% glycerol.
4 Kex2 of embodiment is overexpressed Pichi strain GS115-Kex2 functional verification
Pichi strain GS115-Kex2 is overexpressed to the Kex2 filtered out in embodiment 3 and carries out functional verification.To finish red ferment
Female Expression vector pPIC9K be skeleton by molecular cloning universal method construct respectively containing three sites furin (RXXR, RXRR with
RXKR, X indicate any amino acid) report carrier (attached drawing 3).Report carrier is after the linearisation of Sac I restriction enzyme, by embodiment 2
In method electrotransformation to Kex2 be overexpressed Pichi strain GS115-Kex2 in, with MD plate screening positive transformant.It chooses
The transformant on MD plate is taken to carry out the secreting, expressing of fermentation inducement reporter protein GFP, expression condition presses Invitrogen company
Pichia pastoris operation manual carry out, fermentation supernatant detected with anti-GFP antibody.Processing efficiency finishes with not engineered
Red yeast GS115 is compared (attached drawing 4).
Claims (7)
1. a kind of precursor protein Kex2 is overexpressed Pichi strain, it is characterized in that by Pichia pastoris Kex2 gene, DNA sequence dna
As described in SEQ1, the protein sequence of coding imported into Pichia pastoris after connecting with the expression vector of Pichia pastoris as shown in SEQ2
In, it is built into and Kex2 overexpression Pichi strain.
2. a kind of precursor protein Kex2 is overexpressed Pichi strain construction method, it is characterized in that:
PCR primer design:
1) upstream primer:
Kex2-up: 5'-GGGGAATTCAtgTATTTGCCAGCACTTCGCTTA-3', dashed part areEcoR I restriction enzyme
Restriction enzyme site；
2) downstream primer
Kex2-down:5'-GGGGAATTCTtacaat gccgcacgtttgggatg-3', dashed part areEcoR I limitation
Enzyme restriction enzyme site；
Kex2 gene cloning
1) in superclean bench, one Pichia pastoris GS115 single bacterium of inoculation falls on the conical flask containing 5mLYPD fluid nutrient medium
In, 30 DEG C of culture 24-36h obtain Pichia pastoris culture solution；
2) take 1 ml culture to OD600=1.0~2.0 Pichia pastoris culture solution, 4 DEG C of centrifugation 5min collect thallus；
3) the Pichia pastoris GS115 gene DNA for drawing 100ng ~ 500ng is template, carries out PCR amplification and obtains target gene production
Object；
4) recovery purifying is carried out to target gene product using DNA glue purification QIAquick Gel Extraction Kit, obtains Kex2 gene after purification；
The preparation of Kex2 overexpression Pichi strain
WithEcoR I restriction enzyme respectively to after recovery purifying Kex2 gene and pGAPZB expression plasmid carry out digestion, digestion is complete
Purify the pGAPZB expression plasmid of the Kex2 gene after digestion and linearisation respectively with DNA glue purification QIAquick Gel Extraction Kit afterwards, then
Kex2 gene is connected on pGAPZB expression plasmid using T4 ligase and obtains transformant, transformant connection product is converted big
Kex2 is obtained in enterobacteria TOP 10 is overexpressed Pichi strain.
3. a kind of precursor protein Kex2 according to claim 2 is overexpressed Pichi strain construction method, it is characterized in that:
The PCR amplification condition are as follows: 94 DEG C of 5 min of initial denaturation；It is denaturalized 94 DEG C of 30 sec；Anneal 55 DEG C of 30 sec；Extend 72 DEG C
3 min；30 circulations；72 DEG C of extension 5min.
4. a kind of precursor protein Kex2 according to claim 2 is overexpressed Pichi strain construction method, it is characterized in that:
The transformant send Sangon Biotech (Shanghai) Co., Ltd. to carry out sequencing analysis, and sequence and connection direction are correct
Carrier is named as pGAPZB-Kex2, and the building of Pichi strain is overexpressed for Kex2.
5. a kind of precursor protein Kex2 according to claim 2 is overexpressed Pichi strain construction method, it is characterized in that:
Specific pastoris genomic dna extracts in above-mentioned steps, gene downlink connection and step of converting reference product specification carry out；It is described
T4 ligase, be purchased from TaKaRa company.
6. a kind of precursor protein Kex2 according to claim 2 is overexpressed Pichi strain construction method, it is characterized in that:
The Kex2 gene is as shown in GenBank accession number Accession No CP014716, and the gene order is as described in SEQ1.
7. a kind of precursor protein Kex2 according to claim 2 is overexpressed Pichi strain construction method, it is characterized in that:
The transformant, preparation process are as follows:
1) plasmid linearization, process are: utilizing restriction enzymeAvrIt is linear that II carries out digestion to recombinant plasmid pGAPZB-Kex2
Change, carries out the plasmid pGAPZB-Kex2 after purification and recovery linearisation using DNA glue purification QIAquick Gel Extraction Kit, what is prepared is linear
Change -20 DEG C of plasmid to save backup；
2) Pichia pastoris GS115 feels state cell, and preparation process is: in superclean bench, being inoculated with a Pichia pastoris GS115
Single bacterium is fallen in the 25mL conical flask containing 5mLYPD fluid nutrient medium, 30 DEG C of 12~16h of culture；By the Pichia pastoris of culture
GS115 is transferred according to the ratio of 1:1000 in the big conical flask of 250 mL containing 50mLYPD fluid nutrient medium, and 30 DEG C of cultures 12~
16h, until OD600=1.3~1.5；；Centrifugation, 1500g, 4 DEG C of centrifugation 5min collect thallus, and thallus is sterile with 40mL ice bath
Distilled water is resuspended；Centrifugation: 1500g, 4 DEG C of centrifugation 5min collect thallus, and the aseptic double-distilled water of thallus 25mL ice bath is resuspended；Two
Secondary centrifugation, 1500g, 4 DEG C of centrifugation 5min collect thallus, and the 1M sorbitol solution of thallus 25mL ice bath is resuspended；It is centrifuged again,
1500g, 4 DEG C of centrifugation 5min, collect thallus, 0.5 mL ice bath of thallus 1M sorbitol solution resuspension, Pichia pastoris sense
By state cell；
3) electrotransformation: the linearization plasmid pGAPZB-Kex2 by 5~10 μ g by ice pre-cooling is added to that 80 μ l are above-mentioned to be handled well
Competent cell in, mixing, be added 20 μ l plasmid, mixed liquor is transferred in the conversion cup of preparatory ice bath, is stood on ice
5min；Electrotransformation cup is put into II electric converter of Gene pulser, electricity is carried out by saccharomyces cerevisiae electricity carryover sequence and turns；After electricity turns
The cold 1M sorbitol solution of 1mL ice bath is added into conversion cup immediately, in superclean bench, the yeast weight in cup will be converted
It is outstanding；Conversion fluid is transferred in a 1 new .5ml centrifuge tube, 30 DEG C stationary culture 1 hour；Conversion fluid after drawing culture
200 μ l are coated in the plate of YPD containing Zeocin, 30 DEG C of culture carton upside downs, until transformant occurs；
Picking transformant is inoculated into the 25mL conical flask containing 5mLYPD fluid nutrient medium, 30 DEG C of 24~48 h of culture；It takes
1ml cultivates the Pichia pastoris culture solution to OD600=2.0~2.5, and 1500g, 4 DEG C of centrifugation 5min collect thallus, utilize yeast
RNA extracts reagent agent box by specification operating procedure and extracts total serum IgE.
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| CN105153314A (en) * | 2009-07-09 | 2015-12-16 | 奥普科生物制品有限公司 | Long-acting coagulation factors and methods of producing same |
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| CN105153314A (en) * | 2009-07-09 | 2015-12-16 | 奥普科生物制品有限公司 | Long-acting coagulation factors and methods of producing same |
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| Title |
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| NATHAN C. ROCKWELL 等: "Precursor Processing by Kex2/Furin Proteases", 《CHEM. REV.》 * |
| NCBI: "Komagataella phaffii GS115 Kex2 proprotein convertase (PAS_chr2-1_0304), partial mRNA", 《NCBI REFERENCE SEQUENCE: XM_002491154.1》 * |
| STRAUSBERG,R.L.等: "Homo sapiens furin (paired basic amino acid cleaving enzyme), mRNA (cDNA clone MGC:20463 IMAGE:4550620), complete cds", 《GENBANK: BC012181.1》 * |
| SUMA SREENIVAS等: "Enhancement in production of recombinant two-chain Insulin Glargine by over-expression of Kex2 protease in Pichia pastoris.", 《APPL MICROBIOL BIOTECHNOL》 * |
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