CN105087405A - Pichia pastoris underglycosylated mutant strain and application of pichia pastoris underglycosylated mutant strain - Google Patents
Pichia pastoris underglycosylated mutant strain and application of pichia pastoris underglycosylated mutant strain Download PDFInfo
- Publication number
- CN105087405A CN105087405A CN201510591803.3A CN201510591803A CN105087405A CN 105087405 A CN105087405 A CN 105087405A CN 201510591803 A CN201510591803 A CN 201510591803A CN 105087405 A CN105087405 A CN 105087405A
- Authority
- CN
- China
- Prior art keywords
- mutant strain
- gene
- pichia spp
- hypo
- och1p
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a pichia pastoris underglycosylated mutant strain and application of the pichia pastoris underglycosylated mutant strain to expression of human granulocyte-macrophage colony stimulatory factors. The pichia pastoris underglycosylated mutant strain is obtained through knocking out ochlp genes of pichia pastoris; the nucleotide sequence of the ochlp gene is shown as SEQ ID NO.1; pichia pastoris underglycosylated host bacteria are successfully built; optimized selection is provided for a pichia pastoris system to express recombination protein with multiple glycosylation sites; and the condition that the ochlp genes take part in the pichia pastoris expression protein glycosylation is proved from the angle of molecules. The invention provides the technical foundation and the method guidance for the genetic modification on the pichia pastoris expression host bacteria by using a metabolic engineering technology.
Description
(1) technical field
The present invention relates to a kind of method of foreign protein high expression, particularly a kind of structure of hypo-glycosylated pichia spp mutant strain and the application in expression foreign protein GM-CSF.
(2) background technology
Pichia yeast expression system has become very successfully one of exogenous protein expression system.Much albumen is succeeded recombinant expressed by this system, comprises a lot of industrial enzyme, pharmaceutical protein.Bichi yeast system has many advantages: (1) utilizes alcohol oxidase (alcoholoxidaseI, the AOX1) promotor by methanol induction, strictly can control the expression of foreign gene, expresses a large amount of albumen in the short period of time; (2) pichia spp growth fast culture condition is simple, and be applicable to high-density culture, after fermentation, in often liter of nutrient solution, wet cell weight can reach 450 grams, is conducive to improving target protein output; (3) pichia spp can by signal for locating foreign protein is positioned in peroxysome express or secretion to outside born of the same parents, be therefore applicable to very much expressing the albumen to the toxic effect of host; (4) recombinant vectors is integrating vector, and exogenous origin gene integrator, on yeast chromosomal, keeping selective pressure without the need to adding microbiotic in fermenting process, avoiding the problem of antibiotic remains in recombinant protein.So far existing more than 500 kind of foreign protein successful expression in this expression system, the most high expression level amount reported is respectively 22g/L (in born of the same parents) and 14.8g/L (secretion).
Excessive N-can be carried out to albumen during Pichia anomala expression foreign protein glycosylation modified, produce the high mannose type sugar chain of 12-15 glycosyl, have a strong impact on activity and the immunogenicity of recombinant protein.In this glycosylation process, α-1,6-mannose transferase (och1p) plays keying action in this course.By knocking out α-1,6-mannose transferase (och1p) gene of pichia spp, thus acquisition one has significant application value to the pichia yeast expression system that recombinant protein carries out hypo-glycosylated modification.
(3) summary of the invention
The object of the invention is to provide a kind of hypo-glycosylated mutant strain of pichia spp of hypo-glycosylated expression human granulocyte-macrophage colony stimulating factor and is expressing the application in human granulocyte-macrophage colony stimulating factor, described mutant strain is that α-1,6-mannose transferase (och1p) encoding gene of pichia spp is knocked out structure.
The technical solution used in the present invention is:
The invention provides the hypo-glycosylated mutant strain of a kind of pichia spp, the hypo-glycosylated mutant strain of described pichia spp is obtained by the och1p gene knockout of pichia spp (preferred Pichia pastoris GS115), and the nucleotides sequence of described och1p gene is classified as shown in SEQIDNO.1.
Hypo-glycosylated mutant strain builds pichia spp of the present invention as follows: the linear suicide of (1) och1p gene knocks out plasmid pKO19-och1p: the fragment be in turn connected in order by och1p upstream region of gene flanking sequence (1000bp), pichia spp constitutive promoter pTEF1/EM7, hygromycin gene, terminator CYC1 and och1p downstream of gene flanking sequence (1000bp) be connected on pMD19-T carrier, be built into plasmid pKO19-och1p; The nucleotides sequence of described och1p upstream region of gene flanking sequence is classified as shown in SEQIDNO.2, described promotor pTEF1/EM7 nucleotides sequence is classified as shown in SEQIDNO.4, described hygromycin gene nucleotides sequence is classified as shown in SEQIDNO.5, and described terminator CYC1 nucleotides sequence is classified as shown in SEQIDNO.6; The nucleotides sequence of described och1p downstream of gene flanking sequence is classified as shown in SEQIDNO.3; (2) with plasmid pKO19-och1p for template, under the effect of primer UpF and DownR, carry out pcr amplification, obtain linear knockout dna fragment;
UpF:AACGAAGGAGAATGCCGAAA (shown in SEQIDNO.7);
DownR:CTGAGTTTGGAGGTGCTGTAGGT (shown in SEQIDNO.8);
(3) linear transfor DNA fragmentation step (2) obtained transforms pichia spp (preferred Pichia pastoris GS115), obtain and transform bacterial strain, screening positive clone, obtains the hypo-glycosylated mutant strain of described pichia spp (preferably transforming the mutant strain GS115m that Pichia pastoris GS115 obtains).
Further, the described positive colony screening method of step (3) is: by transforming, the MD of bacterial strain coating containing 50 μ g/ml Totomycin and 10 μ g/ml Histidines is dull and stereotyped, cultivate 3 days, utilize primer HgyF and HgyR to carry out bacterium colony PCR qualification for 30 DEG C, screening obtains positive colony;
HgyF:ATGGAAAAGCCTGAACT (shown in SEQIDNO.9);
HgyR:CTATTTCTTTGCCCTCGGACGA (shown in SEQIDNO.10).
The present invention also provides the hypo-glycosylated mutant strain of a kind of described pichia spp expressing the application in human granulocyte-macrophage colony stimulating factor (GM-CSF), concrete described application is that human granulocyte-macrophage colony stimulating factor encoding gene (shown in SEQIDNO.11) is building up to expression vector pPIC9k, be transformed in the hypo-glycosylated mutant strain of pichia spp after the recombinant plasmid pPIC9k-GM-CSF linearizing obtained, obtain the hypo-glycosylated mutant strain of pichia spp (preferably restructuring GS115m engineering bacteria) containing GM-CSF gene, abduction delivering, obtain the nutrient solution of the hypo-glycosylated human granulocyte-macrophage colony stimulating factor of high expression, by nutrient solution separation and purification, obtain human granulocyte-macrophage colony stimulating factor albumen, i.e. hypo-glycosylated leukine albumen.
Further, the method for the described pichia spp hypo-glycosylated mutant strain abduction delivering GM-CSF albumen containing GM-CSF gene is: the hypo-glycosylated mutant strain of pichia spp containing GM-CSF gene is seeded to YPD substratum, 180rpm, 30 DEG C be cultured to OD
600=1, nutrient solution is forwarded to BMGY substratum, 180rpm, 30 DEG C of cultivations are after 24 hours, hold over night, outwells supernatant after bacterial sediment, precipitation switching BMMY substratum, 180rpm, 30 DEG C of abduction deliverings 72 hours, every 24 hours in Induction Process, add the pure methyl alcohol accounting for the filtration sterilization of nutrient solution cumulative volume 1%, finally obtain the nutrient solution of expressing hypo-glycosylated human granulocyte-macrophage colony stimulating factor.
Further, the method of described nutrient solution separation and purification is: will express the medium centrifugal of human granulocyte-macrophage colony stimulating factor, collect supernatant liquor, get supernatant liquor and add the trichoroacetic acid(TCA) accounting for supernatant volume 1/2, the centrifugal 10min of 12000rpm, abandon supernatant, collecting precipitation, add the Tris-HCl damping fluid of 10mM, pH7.0, suspend precipitation, centrifugal, get supernatant liquor and carry out Ni-NTA affinity chromatography, with the imidazoles aqueous solution wash-out of 100mM, collect elutriant, obtain human granulocyte-macrophage colony stimulating factor albumen.
The linear suicide of och1p gene of the present invention knocks out in plasmid pKO19-och1p structure, by pichia spp constitutive promoter pTEF1/pEM7 (shown in SEQIDNO.4), hygromycin gene (shown in SEQIDNO.5), terminator CYC1 sequence (shown in SEQIDNO.6) is fused into resistance expression's box cassete, expression cassette fragment is again with on och1p gene, downstream flanking sequence (shown in SEQIDNO.2 and SEQIDNO.3) is through overlappingPCR, be fused into a long segment, then this long segment is connected to structure on pMD19-T carrier and forms plasmid pKO19-och1p.PTEF1/EM7 promotor provides strong transcription initiation, and CYC1 sequence provides effective Transcription Termination and polyA to modify signal, and hygromycin gene provides selection markers.
The present invention adopts PCR method to obtain linear knockout dna fragment, and working method is: with plasmid pKO19-och1p for template, under the effect of primer UpF and DownR, carry out pcr amplification, obtains linear transfor DNA fragmentation; Pcr amplification condition is as follows: 94 DEG C of denaturation 51min; 94 DEG C of sex change 50s, 60 DEG C of annealing 50s, 72 DEG C extend 2min, 30 circulations; 25 DEG C of insulation 10min; The present invention adopts electric shocking method linear knockout dna fragment to be converted into pichia spp (preferred GS115 bacterial strain), obtains the hypo-glycosylated mutant strain of pichia spp.
The construction process of expression vector pPIC9k-GM-CSF of the present invention is: be separated and extract test kit with RNA again after collector's peripheral blood mononuclear cell and extract total serum IgE, reverse transcription obtains cDNA.With this cDNA for template, under upstream primer GCF (SEQIDNO.12) and downstream primer GCR (SEQIDNO.13) effect, carry out pcr amplification, pcr amplification condition is, 94 DEG C of denaturation 51min; 94 DEG C of sex change 50s, 60 DEG C of annealing 50s, 72 DEG C extend 2min, 30 circulations; 25 DEG C of insulation 10min; Amplified production cuts the multiple clone site being connected into the pPIC9k carrier cut through same enzyme by EcoRI and Not I enzyme, obtain recombinant plasmid pPIC9K-GM-CSF.
The step that people source of the present invention GM-CSF encoding gene is transformed into the hypo-glycosylated mutant strain of pichia spp (GS115m) is: use primer AOX3FF (SEQIDNO.14) and AOX5RF (SEQIDNO.15), with recombinant plasmid pPIC9K-GM-CSF for template, the method of PCR is adopted to carry out linearizing to plasmid, thus obtain with G418 resistance expression box, the linear DNA fragment of Histidine synthetic enzyme and GM-CSF expression cassette, then this DNA fragmentation is made to transform the hypo-glycosylated mutant strain of pichia spp by electroporated method, thalline suspension is coated on MD flat board, every 200-600 μ l is coated with one flat plate, flat board is placed in 30 DEG C of cultivations, until single bacterium colony occurs, obtains the hypo-glycosylated mutant strain of pichia spp (i.e. the positive recombinant bacterium of GS115m) containing GM-CSF gene.
The method of the pichia spp hypo-glycosylated mutant strain bacterium colony PCR containing GM-CSF gene of the present invention and high copy colony screening is:
Use upstream primer GCF and downstream primer GCR, direct bacterium colony PCR, the positive recombinant bacterium bacterium colony of the hypo-glycosylated mutant strain of pichia spp containing GM-CSF gene is determined in screening.Pcr amplification condition is, 94 DEG C of denaturation 51min; 94 DEG C of sex change 50s, 60 DEG C of annealing 50s, 72 DEG C extend 2min, 30 circulations; 25 DEG C of insulation 10min.Positive recombinant bacterium bacterium colony is dull and stereotyped with the MD of different concns G418 antibiotics resistance (0.1,0.2,0.5,1,2,3,4,5mg/ml) with toothpick dibbling.The restructuring bacterium colony grown by screening the microbiotic of resistance to higher concentration, thus obtain the recombinant bacterial strain carrying high copy people source GM-CSF gene on genome.
High copy recombinant bacterial strain carries out fermentation culture, obtains the nutrient solution of high expression hypo-glycosylated people source GM-CSF albumen.Fermentation culture conditions is as follows:
Recombinant bacterial strain list colony inoculation YPD substratum, 180rpm, 30 DEG C cultivate 24 little of OD600nm=1, switching BMGY substratum, 180rpm, 30 DEG C of cultivations are after 24 hours, hold over night, outwells supernatant after yeast sedimentation, then adds BMMY substratum, 180rpm, 30 DEG C of abduction deliverings 72 hours, obtain the nutrient solution of high expression hypo-glycosylated people source GM-CSF albumen.
The collection of people source of the present invention GM-CSF albumen, analysis and purification process are: the medium centrifugal of hypo-glycosylated people source GM-CSF albumen is collected supernatant liquor, get 500 RI of supernatant, add 50 microlitre 100% trichoroacetic acid(TCA)s, the centrifugal 10min of 12000rpm, abandon supernatant, collecting precipitation, adds 50 microlitre 10mMTrisHCl (pH7.0) damping fluids, and suspend precipitation, get 10 microlitres, carry out SDS-PAGE electrophoretic analysis expression of recombinant proteins situation.Supernatant liquor will be remained by Ni-NTA affinity chromatography (affinity column is Qiagen company Ni-NTAAgarose, catnumber30210), with the imidazoles aqueous solution wash-out of 100mM, obtain purifying protein.Then purity and the content of purifying protein is detected with SDS-PAGE.The rhGM-CSF albumen that the hypo-glycosylated mutant strain of the pichia spp that the present invention builds is expressed, degree of glycosylation is only 22%, and the GM-CSF protein glycosylation degree that original strain is expressed reaches 50%.
BMGY substratum quality of the present invention consists of: yeast extract 1%, peptone 2%, yeast nitrogen (YNB) 1.34%, vitamin H (4 × 10
-5) %, glycerine 1%, yeast nitrogen and vitamin H filtration sterilization, solvent is 100mM potassium phosphate buffer (pH6.0), and pH value is 6.0.
BMMY substratum quality of the present invention consists of: yeast extract 1%, peptone 2%, yeast nitrogen (YNB) 1.34%, vitamin H (4 × 10
-5) %, methyl alcohol 3%, yeast nitrogen and vitamin H filtration sterilization, solvent is 100mM potassium phosphate buffer (pH6.0), and pH value is 6.0.
The dull and stereotyped quality group of MD of the present invention becomes: yeast nitrogen (YNB) 1.34%, vitamin H (4 × 10
-5) %, glucose 2%, solvent is water, and pH value is 6.0.
YPD substratum quality of the present invention consists of: peptone 2%, yeast extract 1%, glucose 2%, and solvent is water, and pH value is 6.0.
The quality composition of YPD selectivity flat board: 2% peptone, 1% yeast extract, 2% glucose, 1.5% agar powder, solvent is water, pH value 6.0.
Compared with prior art, beneficial effect of the present invention is mainly reflected in: the present invention has cloned α-1,6-mannose transferase (och1p) gene and the upstream and downstream flanking nucleotide sequence thereof that participate in protein glycosylation first from pichia spp.With pichia spp Host Strains GS115 for operand, adopt linear suicide vector, electroporated, the mode of resistance screening, has knocked out α-1,6-mannose transferase (och1p) gene first in pichia spp.Obtain the hypo-glycosylated mutant strain GS115m of pichia spp of hypo-glycosylated ability.Utilize genetic engineering technique high expression level people source GM-CSF albumen in the hypo-glycosylated mutant strain GS115m of pichia spp first.The present invention successfully builds the hypo-glycosylated Host Strains of pichia spp, expresses providing with polyglycosylated site recombinant protein the selection more optimized for Bichi yeast system.The present invention proves α-1 from molecule angle, 6-mannose transferase (och1p) gene take part in the glycosylation of Pichia anomala expression albumen, and the present invention provides technical foundation and guide for method for utilizing metabolic engineering technology to carry out genetic modification to Pichia anomala expression Host Strains.
(4) accompanying drawing explanation
Fig. 1 is α-1,6-mannose transferase (och1p) gene and upstream and downstream flanking sequence pcr amplification product agarose electrophoresis figure thereof, M1:DNA molecular weight standard (DL2000), M:DNA molecular weight standard (λ/HindIIIDNAMarker), 1:och1p upstream region of gene flanking sequence, 2:och1p gene, 3:och1p downstream of gene flanking sequence, 4:GS115 strain gene group DNA, 5: resistance expression's box cassete.
Fig. 2 is with the pichia spp suicide vector pKO19-och1p collection of illustrative plates of och1p gene upstream and downstream flanking sequence.
The agarose electrophoresis figure of the linear knockout dna fragment of Fig. 3, swimming lane M is DNAmarkerD2000, and swimming lane 1-6 is 6 Duplicate Samples that the amplification of restructuring plasmid PCR knocks out fragment.
Fig. 4 GS115m bacterial strain transforms bacterium colony PCR electrophorogram, M representation DNA molecular weight markerD2000, and 1-6 swimming lane represents the bacterium colony PCR primer of 6 different yeast clones.
The SDS-PAGE collection of illustrative plates of Fig. 5 original strain and mutant strain expression of GM-csf protein.M represents molecular weight of albumen marker, and 1-3 swimming lane represents the outer total protein (containing object band) of born of the same parents that original strain 3 transformed clones are expressed, and 4-6 swimming lane represents the outer total protein (containing object band) of born of the same parents that mutant strain 3 transformed clones are expressed.
The mensuration of Fig. 6 original strain and the outer total protein content of mutant strain born of the same parents.
Fig. 7 purification of Recombinant GM-CSF albumen and N Glycosylase F enzyme thereof cut rear SDS-PAGE collection of illustrative plates, M represents molecular weight of albumen marker, 1 swimming lane represents the recombinant protein that original strain is expressed, 2 swimming lanes represent N-Glycosylase F enzyme cut process after original strain expressing protein, 3 swimming lanes represent the recombinant protein that mutant strain is expressed, 4 swimming lanes represent N-Glycosylase F enzyme cut process after mutant strain expressing protein, 5 swimming lanes represent the GM-CSF of commercial Recombinant protein expression.
(5) embodiment
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in this:
Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described in detail.The experimental technique of unreceipted actual conditions in embodiment, usually conveniently condition, the such as condition described in Molecular Cloning: A Laboratory guide (third edition, the work such as J. Pehanorm Brooker), or according to the condition that manufacturer advises.
The present invention passes through bioinformatic analysis, according to the genomic information of pichia pastoris phaff, determine α-1,6-mannose transferase (och1p) gene and upstream and downstream flanking sequence information thereof, electricity is adopted to transform, the steps such as homologous recombination have knocked out this gene, obtain mutant yeast strain GS115m.Choose foreign gene carries out recombinant expressed in GS115m and GS115, compares molecular weight and the degree of glycosylation of recombinase, summarizes using value and the prospect of mutant strain GS115m.The clone of the flanking gene pack section of embodiment 1, each 1000bp of pichia spp α-1,6-mannose transferase (och1p) upstream and downstream
The α-1 of the Pichia pastoris GS115 bacterial strain utilizing bioinformatic analysis to search to report in NCBI, 6-mannose transferase (och1p) gene, obtain gene flank upstream and downstream sequence information (Genbanknumber), use primer5.0 software design primer, upstream primer is: och1upF:5 '-3 ' (SEQIDNO.16), och1upR:5 '-3 ' (SEQIDNO.17); Downstream primer is: och1downF:5 '-3 ' (SEQIDNO.18), och1downR:5 '-3 ' (SEQIDNO.19).With yeast total DNA extraction test kit (purchased from BIOSCI company, article No. is RT001) (open source literature is originated: NatureBiotechnology27 to extract Pichia pastoris GS115,561-566 (2009), Publishedonline:24May2009doi:10.1038/nbt.1544) STb gene, and as template, carry out pcr amplification.
Amplification condition is: 94 DEG C of denaturation 5min; Circulate interior 94 DEG C of sex change 30sec, 50 DEG C of annealing 30sec, and 68 DEG C extend 30S, 32 circulations; 10min is extended after 68 DEG C.Two PCR primer, through agarose electrophoresis, reclaim object fragment respectively, after recovery product adds the process of A test kit, reclaim fragment and connect TVector, transform JM109 competent cell, send order-checking after screening positive clone.Positive colony plasmid called after pMD18-och1up and pMD18-och1down, be with the Insert Fragment just like nucleotide sequence shown in SEQIDNO.2 and SEQIDNO.3, be designated as (och1p) gene upstream and downstream flanking fragment, obtain plasmid pMD18-och1up and plasmid pMD18-och1down respectively.
The structure of embodiment 2och1p enzyme gene knockout carrier pKO19-och1P
(1) structure of hygromycin expression cassette
TEF-EF7F：TATTGTGTTGTAGGAGCCCACACACCATAGC；
TEF-EF7R：AGTTCAGGCTTTTCCATGGTTTAGTTCCTCACC；
HgyTYF：5’-GGTGAGGAACTAAACCATGGAAAAGCCTGAACTCA-3’(SEQIDNO.9)；
HgyTYR：5’-TCGGACGTGAAGCTTCTATTTCTTTGCCCTCG-3’(SEQIDNO.10)；
CYC1F：CGAGGGCAAAGAAATAGAAGCTTCACGTCCGA；
CYC1R：ATAACTTTACTATTTACAAATTAAAGCCTT；
TEF-EF7F/R primer pair and CYC1F/R all with pGAPZ α A plasmid (purchased from Invitrogen company) for template carries out pcr amplification, HgyTYF/R primer pair with pcambia1300 plasmid (purchased from cambia company) for template carries out pcr amplification.
Pcr amplification condition is: 94 DEG C of denaturation 5min; 94 DEG C of sex change 50s, 59 DEG C of annealing 50s, 68 DEG C extend 1min, 32 circulations; 5min is extended after 68 DEG C; 25 DEG C of insulation 10min.The amplified production of above-mentioned three PCR reaction is reclaimed test kit with sepharose DNA product respectively reclaim.
Above-mentioned three PCR primer, as template, with TEF-EF7F and CYC1R primer, carry out OverlappingPCR reaction.Pcr amplification condition is: 94 DEG C of denaturation 5min; 94 DEG C of sex change 50s, 59 DEG C of annealing 50s, 68 DEG C extend 3min, 32 circulations; 5min is extended after 68 DEG C; 25 DEG C of insulation 10min.PCR primer is for subsequent use after reclaiming, called after Hgycassete fragment.
(2) with embodiment 1 synthesize pMD18-och1up and pMD18-och1down plasmid for template, with upstream primer be: och1upF:5 '-3 ' (SEQIDNO.16), och1upR:5 '-3 ' (SEQIDNO.17); Downstream primer is: och1downF:5 '-3 ' (SEQIDNO.18), och1downR:5 '-3 ' (SEQIDNO.19).Carry out pcr amplification respectively, for subsequent use after PCR primer purifying, called after upstream flanking fragment TYup (SEQIDNO.2) and downstream flanking fragment TYdown (SEQIDNO.3).
With Hgycassete fragment, upstream flanking fragment TYup and downstream flanking fragment TYdown, 3 DNA fragmentations are template, och1upF and och1downR is primer, carries out overlappingPCR, and pcr amplification condition is: 94 DEG C of denaturation 5min; 94 DEG C of sex change 50s, 59 DEG C of annealing 50s, 68 DEG C extend 5min, 32 circulations; 5min is extended after 68 DEG C; 25 DEG C of insulation 10min.PCR primer reclaims, and after recovery product adds the process of A test kit, reclaims fragment and connects TVector, transform JM109 competent cell, send order-checking after screening positive clone.Positive colony plasmid called after plasmid pKO19-och1P.
The structure of embodiment 3 pichia spp deficient strain
1. the preparation of linear transfor fragment
With pKO19-och1P plasmid for template, UpF and DownR is that primer carries out pcr amplification, and after PCR primer purifying, concentration quantitative is 100ng/ μ l.
UpF：AACGAAGGAGAATGCCGAAA(SEQIDNO.7)；
DownR：CTGAGTTTGGAGGTGCTGTAGGT(SEQIDNO.8)；
2. pichia spp electricity transforms
(1) with transfering loop picking one ring Pichia pastoris GS115 list bacterium colony, be seeded in the 50ml triangular flask containing 5mlYPD substratum, 30 DEG C, 250-300rpm/min overnight incubation;
(2) be forwarded in the 2L triangle shaking flask containing 500ml fresh YPD medium by step (1) culture 100-500 μ l, 28-30 DEG C, 250-300rpm/min overnight incubation, to OD
600reach 1.3-1.5;
(3) by step (2) culture in 4 DEG C, the centrifugal 5min of 1500g, with the sterilized water of the ice precooling of 500ml, bacterial sediment is resuspended;
(4) by centrifugal for step (3) bacteria suspension, outwell supernatant, then use the sterilized water of the ice precooling of 250ml that bacterial sediment is resuspended;
(5) by centrifugal for step (4) bacteria suspension, outwell supernatant, then use the sorbitol aqueous solution of the 1M of the ice precooling of 20ml that bacterial sediment is resuspended;
(6) by centrifugal for step (5) bacteria suspension, outwell supernatant, then use the sorbitol aqueous solution of the 1M of the ice precooling of 1ml that bacterial sediment is resuspended, obtain thalline suspension, thalline suspension final volume is about 1.5ml;
(7) be packed as the packaging of 80 μ l portions ,-80 degree are preserved.
Electroporated:
(8) electricity is transformed cup ice bath 5min;
(9) by the linearizing DNA (TE solution, volume is about 5 ~ 10 μ l) of 5-20 μ g, mix with the thalline suspension of above-mentioned steps (6) gained of 80 μ l, the electricity going to the precooling of 0.2cm ice transforms cup;
(10) Studies on Electroporation Transformation: voltage 1.5kV; Electric capacity 25 μ F; Resistance 200 Ω.The electric shock time is 4-10 millisecond.
(11), after electric shock, thalline mixes by the sorbitol aqueous solution adding the 1M of 1ml ice precooling, goes in the EP pipe of 1.5ml;
(12) coated by thalline suspension on YPD (40ng/ μ l Totomycin) flat board, every 200-600 μ l is coated with one flat plate;
(13) flat board is placed in 30 DEG C of cultivations, until single bacterium colony occurs.
3. positive colony screening
(1) the positive bacterium colony of toothpick picking is cloned in PCR pipe, adds in 10 ul sterile water the thalline that suspends, and be placed in PCR instrument 98 degree process 10min, the then centrifugal 5min of 1000rpm, supernatant is transferred in clean PCR pipe.
(2) 2 RI of supernatant of step (1) are got, with och1upF (SEQIDNO.16) and HgyTYR (SEQIDNO.10) for primer carries out pcr amplification, agarose gel electrophoresis detects PCR primer size, can amplify the clone of 2.6kb clip size for positive.
(3) positive colony uses YPD liquid nutrient medium (40 μ g/ml Totomycin), 30 degree, 180rpm incubated overnight, the bacterium colony of normal growth can reaffirm positive bacterium colony, obtain the hypo-glycosylated mutant strain of pichia spp, be designated as bacterial strain GS115m.
Embodiment 4 pichia spp hypo-glycosylated mutant strain GS115m expression and purification recombination human source GM-CSF Protein Assav
1, the construction step of expression vector pPIC9k-GM-CSF:
(1) human peripheral mononuclear leukocyte extracts test kit with RNA and extracts total serum IgE, and reverse transcription obtains cDNA.
(2) with this cDNA for template, upstream primer GCF (SEQIDNO.12) and downstream primer GCR (SEQIDNO.13) effect carry out pcr amplification, pcr amplification condition is, 94 DEG C of denaturation 5min; 94 DEG C of sex change 50s, 60 DEG C of annealing 50s, 72 DEG C extend 2min, 30 circulations; 25 DEG C of insulation 10min.
(3) cut by EcoRI and Not I enzyme the multiple clone site being connected into the pPIC9k carrier cut through same enzyme after PCR primer purifying, transform JM109 competent cell, after screening, obtain recombinant plasmid pPIC9k-GM-CSF.
2, recombinant plasmid pPIC9k-GM-CSF is transformed into the step of the hypo-glycosylated mutant strain GS115m of pichia spp and Pichia pastoris GS115:
(1) primer AOX3FF and AOX5RF (SEQIDNO.14 and SEQIDNO.15), with recombinant plasmid pPIC9K-GM-CSF for template, the method for employing PCR obtains the conversion linear DNA fragment with Histidine synthetic enzyme expression cassette and GM-CSF expression cassette.
(2) carry out, except following change according to the step that in embodiment 3, pichia spp electricity transforms and positive colony screens:
1) needed for the coating in step (12), flat board is replaced by MD flat board, and the dull and stereotyped quality group of MD becomes: 1.34%YNB, 4 × 10
-5% vitamin H, 2% glucose, 1.5% agar powder, solvent is water, pH value 6.
2) primers designed och1upF and HgyTYR changes the primer of bacterium colony PCR qualification, upstream primer GCF and downstream primer GCR (SEQIDNO.12 and SEQIDNO.13), can amplify the clone of 370bp clip size for positive.
3) Host Strains being used for transforming has GS115 and GS115m two kinds, transforms simultaneously, screening and induction.
3, high copy positive colony screening
Preparation is containing different concns G418 microbiotic (0.5,1,1.5,2,2.5,3,3.5,4,4.5,5mg/ml) MD dull and stereotyped, by the dibbling of step 2 positive colony, select the positive colony bacterial strain of the above G418 of tolerance 4mg/ml concentration, be defined as high copy positive colony--leukine Pichia yeast engineering.
4, leukine Pichia yeast engineering fermentation culture step
(1) by leukine Pichia yeast engineering list colony inoculation 5mlYPD substratum, 180rpm, 30 degree of cultivations 24 hours, OD
600=1.
(2) get step (1) nutrient solution 5ml to transfer 100mlBMGY substratum, 180rpm, 30 degree of cultivations 24 hours.Cultivate after 24 hours, hold over night, supernatant BMGY substratum is outwelled after yeast sedimentation, add 500mlBMMY substratum again, 30 DEG C of abduction deliverings 72 hours, between inductive phase, every 24 hours, add the pure methyl alcohol of filtration sterilization of nutrient solution cumulative volume 1%, finally obtain leukine Pichia yeast engineering fermented liquid.
BMGY substratum quality consists of: yeast extract 1%, peptone 2%, yeast nitrogen (YNB) 1.34%, vitamin H (4 × 10
-5) %, glycerine 1%, YNB and vitamin H filtration sterilization, solvent is 100mM potassium phosphate buffer (pH6.0).
BMMY substratum quality consists of: yeast extract 1%, peptone 2%, yeast nitrogen (YNB) 1.34%, vitamin H (4 × 10
-5) %, YNB and vitamin H (Biotin) filtration sterilization, solvent is 100mM potassium phosphate buffer (pH6.0).
(3) by step (2) fermented liquid collected by centrifugation supernatant liquor.Get 500 RI of supernatant, add 50 microlitre 100%TCA, the centrifugal 10min of 12000rpm, abandon supernatant, collecting precipitation, adds 50 microlitre 10mMTris-HCl (pH7.0) damping fluids, and suspend precipitation, get 10 microlitres and utilize the outer total protein content of Xylene Brilliant Cyanine G (Bradford) method determination of protein concentration kit measurement born of the same parents, the results are shown in Figure 5; Separately get 10 microlitres, carry out SDS-PAGE electrophoretic analysis expression of recombinant proteins situation, the results are shown in Figure 6.
According to the outer total protein content measurement result of born of the same parents in Fig. 5, and target protein electrophoretic band gray scale measuring and calculating in Fig. 6, the GM-SCF recombinant protein content that original strain and mutant strain are expressed can account for more than 80% of the outer total protein ratio of born of the same parents, the outer total protein content of born of the same parents is about 110mg/L fermented liquid, and target protein output is about 90mg/L fermented liquid.
As can be seen from Fig. 6 result, the expression amount of the leukine that original strain and mutant strain are expressed does not have notable difference, and the leukine molecular weight that original strain and mutant strain are expressed has notable difference.
The object recombinant protein molecular weight that original strain is expressed is about 29KDa, and the object recombinant protein molecular weight that mutant strain is expressed is about 24KDa.This molecular weight of albumen illustrating that hypo-glycosylated mutant strain is expressed has obvious decline, proves that recombinant protein degree of glycosylation has reduction.
5, leukine protein purification
(1) by step 4 (2) leukine Pichia yeast engineering fermented liquid, 10000rpm, 4 DEG C of centrifugal 10min, collect supernatant liquor, 0.45 μm of membrane filtration.
(2) (affinity column is Qiagen company Ni-NTAAgarose to Ni-NTA gel, catnumber30210), use 20mMTris-HCl damping fluid (pH6.5) to balance 2 times, add equal-volume bindingbuffer, make 50% suspension.
(3) every 1ml suspension adds 4ml step (1) supernatant liquor, and 1h is hatched in 30 degree of low speed concussions.
(4), after leaving standstill, suck supernatant liquor, gel 20mMTris-HCl damping fluid (pH6.5) washs 2 times.
(5) add each 20ml of 50mM, 100mM, 500mM imidazoles aqueous solution successively, collect elutriant, 4 degree of dialyzed overnight removing imidazoles, dialyzate is 20mMTris-HCl damping fluid (pH6.5).
(6) the protein sample SDS-PAGE after dialysis detects purity of protein, is merged by the GM-CSF protein sample of the purifying of not assorted band.
6. leukine albumen N-Glycosylase F restriction analysis
(1) get 10 micro liter purified GM-CSF, join in 100 microlitre 0.5%SDS and 0.04mol/LDTT denaturation buffer, 100 DEG C of sex change 10min,
(2) add 100 microlitres again and contain 1%NP-40, in 0.05mol/L sodium phosphate buffer (pH7.2) system of the N-Glycosylase F of 10 units, 37 DEG C of enzymes cut more than 1h,
(3) digestion products carries out SDS-PAGE analysis, the results are shown in Figure 7.
Can be found by the result of Fig. 7, leukine molecular weight after N-Glycosylase F enzyme is cut of original Pichia anomala expression have decreased to 18KDa by 29KDa, have dropped 11KDa.Leukine molecular weight after N-Glycosylase F enzyme is cut of sudden change Pichia anomala expression drops to 18KDa by 24KDa, have dropped 6KDa.The molecular weight declined is exactly the molecular weight of glycosylation sugar chain, and therefore we may safely draw the conclusion, and the leukine of sudden change Pichia anomala expression contains less sugar base sugar chain, has lower level of glycosylation.
By Fig. 7 result we also can learn, the GM-CSF albumen of Recombinant protein expression, without any glycosylation composition, molecular weight is 14KDa.The above results illustrates that the leukine albumen that hypo-glycosylated pichia spp mutant strain is expressed also contains partial glycosylation.
Claims (7)
1. the hypo-glycosylated mutant strain of pichia spp, it is characterized in that the hypo-glycosylated mutant strain of described pichia spp is obtained by the och1p gene knockout of pichia spp, the nucleotides sequence of described och1p gene is classified as shown in SEQIDNO.1.
2. the hypo-glycosylated mutant strain of pichia spp as claimed in claim 1, it is characterized in that the hypo-glycosylated mutant strain of described pichia spp builds as follows: the linear suicide of (1) och1p gene knocks out plasmid pKO19-och1p: be connected on pMD19-T carrier by the fragment connected to form successively by och1p upstream region of gene flanking sequence, pichia spp constitutive promoter pTEF1/EM7, hygromycin gene, terminator CYC1 and och1p downstream of gene flanking sequence, build plasmid pKO19-och1p; Described promotor pTEF1/EM7 nucleotides sequence is classified as shown in SEQIDNO.4, and described hygromycin gene nucleotides sequence is classified as shown in SEQIDNO.5, and terminator CYC1 nucleotides sequence is classified as shown in SEQIDNO.6; The nucleotides sequence of och1p upstream region of gene flanking sequence is classified as shown in SEQIDNO.2, and the nucleotides sequence of described och1p downstream of gene flanking sequence is classified as shown in SEQIDNO.3; (2) with plasmid pKO19-och1p for template, under the effect of primer UpF and DownR, carry out pcr amplification, obtain linear transfor DNA fragmentation;
UpF：AACGAAGGAGAATGCCGAAA；
DownR：CTGAGTTTGGAGGTGCTGTAGGT；
(3) linear transfor DNA fragmentation step (2) obtained transforms pichia spp, and screening positive clone, obtains the hypo-glycosylated mutant strain of described pichia spp.
3. the hypo-glycosylated mutant strain of pichia spp as claimed in claim 2, it is characterized in that the described positive colony screening method of step (3) is: the MD of bacterial strain coating containing 50 μ g/ml Totomycin and 10 μ g/ml Histidines is dull and stereotyped by transforming, cultivate 3 days for 30 DEG C, utilize primer HgyF and HgyR to carry out bacterium colony PCR qualification, obtain positive colony;
HgyF：ATGGAAAAGCCTGAACT；
HgyR：CTATTTCTTTGCCCTCGGACGA。
4. the hypo-glycosylated mutant strain of pichia spp described in a claim 1 is expressing the application in human granulocyte-macrophage colony stimulating factor.
5. apply as claimed in claim 4, it is characterized in that described application is that human granulocyte-macrophage colony stimulating factor encoding gene is building up to expression vector pPIC9k, be transformed in the hypo-glycosylated mutant strain of pichia spp after the recombinant plasmid pPIC9k-GM-CSF linearizing obtained, obtain the hypo-glycosylated mutant strain of pichia spp containing GM-CSF gene, abduction delivering, obtain the nutrient solution of expressing human granulocyte-macrophage colony stimulating factor, by nutrient solution separation and purification, obtain human granulocyte-macrophage colony stimulating factor albumen.
6. apply as claimed in claim 5, it is characterized in that described derivational expression method is: be seeded to YPD substratum by containing the hypo-glycosylated mutant strain of pichia spp of GM-CSF gene, 180rpm, 30 DEG C be cultured to OD
600=1, nutrient solution is forwarded to BMGY substratum, 180rpm, 30 DEG C of cultivations are after 24 hours, hold over night, outwells supernatant after bacterial sediment, precipitation switching BMMY substratum, 180rpm, 30 DEG C of abduction deliverings 72 hours, obtain the nutrient solution of expressing human granulocyte-macrophage colony stimulating factor.
7. apply as claimed in claim 5, it is characterized in that the method for described nutrient solution separation and purification is: the medium centrifugal of human granulocyte-macrophage colony stimulating factor will be expressed, collect supernatant liquor, get supernatant liquor and add the trichoroacetic acid(TCA) accounting for supernatant volume 1/2, the centrifugal 10min of 12000rpm, abandon supernatant, collecting precipitation, add 10mM, the Tris-HCl damping fluid of pH7.0, suspend precipitation, centrifugal, get supernatant liquor and carry out Ni-NTA affinity chromatography, with the imidazoles aqueous solution wash-out of 100mM, collect elutriant, obtain human granulocyte-macrophage colony stimulating factor albumen.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510591803.3A CN105087405A (en) | 2015-09-17 | 2015-09-17 | Pichia pastoris underglycosylated mutant strain and application of pichia pastoris underglycosylated mutant strain |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510591803.3A CN105087405A (en) | 2015-09-17 | 2015-09-17 | Pichia pastoris underglycosylated mutant strain and application of pichia pastoris underglycosylated mutant strain |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105087405A true CN105087405A (en) | 2015-11-25 |
Family
ID=54568741
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510591803.3A Pending CN105087405A (en) | 2015-09-17 | 2015-09-17 | Pichia pastoris underglycosylated mutant strain and application of pichia pastoris underglycosylated mutant strain |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105087405A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110590939A (en) * | 2019-09-20 | 2019-12-20 | 广州暨南大学医药生物技术研究开发中心 | Method for obtaining recombinant human fibronectin by using genetic engineering |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101195809A (en) * | 2006-12-07 | 2008-06-11 | 中国人民解放军军事医学科学院生物工程研究所 | Pichia pastoris strain with deletion of alpha-1,6-mannose transferase and construction method thereof |
| CN102120967A (en) * | 2010-12-09 | 2011-07-13 | 江南大学 | Preparation and application of OCH1 genetic flaw type P. pastoris X-33 bacterial strain |
| WO2011106389A1 (en) * | 2010-02-24 | 2011-09-01 | Merck Sharp & Dohme Corp. | Method for increasing n-glycosylation site occupancy on therapeutic glycoproteins produced in pichia pastoris |
-
2015
- 2015-09-17 CN CN201510591803.3A patent/CN105087405A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101195809A (en) * | 2006-12-07 | 2008-06-11 | 中国人民解放军军事医学科学院生物工程研究所 | Pichia pastoris strain with deletion of alpha-1,6-mannose transferase and construction method thereof |
| WO2011106389A1 (en) * | 2010-02-24 | 2011-09-01 | Merck Sharp & Dohme Corp. | Method for increasing n-glycosylation site occupancy on therapeutic glycoproteins produced in pichia pastoris |
| CN102120967A (en) * | 2010-12-09 | 2011-07-13 | 江南大学 | Preparation and application of OCH1 genetic flaw type P. pastoris X-33 bacterial strain |
Non-Patent Citations (3)
| Title |
|---|
| PIETER P JACOBS ET.AL.: "Engineering complex-type N-glycosylation in Pichia pastoris using GlycoSwitch technology", 《NATURE PROTOCOLS》 * |
| 宋淼等: "重组人粒细胞－巨噬细胞集落刺激因子在毕赤酵母中的表达及活性分析", 《生物技术通讯》 * |
| 张大成等: "毕赤酵母X-33OCH1基因敲除菌株的构建及其表达低糖基化蛋白GM-CSF", 《微生物学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110590939A (en) * | 2019-09-20 | 2019-12-20 | 广州暨南大学医药生物技术研究开发中心 | Method for obtaining recombinant human fibronectin by using genetic engineering |
| CN110590939B (en) * | 2019-09-20 | 2024-02-02 | 广州暨南大学医药生物技术研究开发中心有限公司 | Method for obtaining recombinant human fibronectin by using genetic engineering |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102839165B (en) | Gene mutation type recombined protease K and industrialized production method thereof | |
| JPH0543348B2 (en) | ||
| CN103937830B (en) | A kind of recombinant bacterium of efficient secretory expression Nattokinase | |
| CN105358706A (en) | Yeast promoters for protein expression | |
| CN113106114A (en) | Factor for regulating and controlling trichoderma reesei protein expression efficiency, regulating and controlling method and application | |
| CN112522125B (en) | Hyaluronidase engineering bacterium and construction method and application thereof | |
| JPWO2020027010A1 (en) | Method for producing mutant strain and protein of Trichoderma filamentous fungus | |
| CN105087405A (en) | Pichia pastoris underglycosylated mutant strain and application of pichia pastoris underglycosylated mutant strain | |
| CN103114089B (en) | Strong promoter from trichoderma reesei as well as expression vector and application thereof | |
| JPH08500014A (en) | K. Promoter of K. lactis transaldolase gene and use thereof | |
| CN102533808B (en) | Method for expressing and purifying recombinant ethanol oxidase in pichia pastoris | |
| CN103374570B (en) | Promoter of filamentous fungus glyceraldehyde-3-phosphate dehydrogenase gene (gpd) and application of promoter | |
| JPH08500007A (en) | K. Promoter of the K. lactis pyruvate decarboxylase gene and its use | |
| CN105755035B (en) | A kind of building of Hansenula yeast specific expression vector and hepatitis B virus surface antigen is being improved in the method for expressed by Hansenula yeast amount | |
| CN111518851B (en) | Immobilized enzyme continuous preparation 14/15 N]Process for preparing L-citrulline | |
| AU2020204574B2 (en) | B.licheniformis engineering bacteria for inhibiting bacterial autolysis, construction method and application thereof | |
| CN108342400B (en) | Genetic engineering bacterium for recombinant expression of oxalate oxidase and construction method and application thereof | |
| JP2003509054A (en) | Regulatory sequences and expression cassettes for yeast | |
| JPH06311884A (en) | Plasmid and escherichia coli transformed with the same | |
| CN117050163B (en) | Pichia pastoris engineering bacteria for secretory expression of recombinant type III collagen and application thereof | |
| CN110527690A (en) | A kind of heat resistant type tannase and its application | |
| CN113293153B (en) | Method for secretory expression of levansucrase by recombinant pichia pastoris and application thereof | |
| CN110616209A (en) | Mutant RNAseA and expression and purification method thereof in yeast cells | |
| CN117343950B (en) | Method for improving expression level of trichoderma reesei expression exogenous protein | |
| EP0248227A1 (en) | Yeast production of streptokinase |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20151125 |