CN103966253A - Method for efficiently preparing recombinant human interluekin-33 protein - Google Patents
Method for efficiently preparing recombinant human interluekin-33 protein Download PDFInfo
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Abstract
The invention relates to a recombinant vector, method and recombinant pichia pastoris engineering strain for efficiently preparing a recombinant human interluekin-33 (rhIL33-Fc) protein and a produced recombinant protein. The method comprises the following steps of (1) constructing an expression vector of a human interluekin-33 protein, wherein the expression vector contains coded sequences of an expression regulation element, a secretion signal peptide, human interluekin-33 and an IgG1-Fc fusion protein; (2) constructing and screening an efficient expression strain of pichia pastoris of the human interluekin-33 protein; (3) optimizing the fermentation pH value, temperature and carbon source of the engineering strain; and (4) separating, purifying and physicochemically authenticating the human interluekin-33 protein. By using the method provided by the invention, the recombinant human interluekin-33 protein with an industrial value can be efficiently obtained.
Description
Technical field
The invention belongs to technical field of bioengineering, be specifically related to method, the Pichia yeast engineering of high efficient expression rhIL33-Fc and expressed recombinant protein with pichia spp Restruction Ro 24-7472/000-33 albumen (rhIL33-Fc).
Background technology
Interleukin 33 (IL-33) is the member of IL-1 family, and it and IL-1 β and IL-18 are similar, in the immune response of body, play an important role.IL-33 is the albumen of a 30kDa, more conservative in evolution, and the sequence in the sequence in people source and mouse source has 54% homology.Normal IL-33 is mainly expressed in endotheliocyte and epidermic cell, and its release is often accompanied by the damage of body or the necrosis of cell, thereby it is also considered to a kind of warning albumen (Alarmin).IL-33 is by being combined and recruiting MyD88 with the acceptor ST2 of cell surface, IRAK1/4 and TRAF6, and then cause the activation of NF-κ B and the release of the proinflammatory inflammation factor of Th-2, and as IL-4, the factors such as IL-5 and IL-13.The IL-33 being discharged in blood is conducive to promote body defence parasite, and bacterium infects and virus infection, and even it is also considered to have the myocardium function of protection.
Before the present invention, recombinant human interleukin-33 (rhIL-33) of selling on market are mainly to use escherichia coli expression and preparation.As everyone knows, albumen recombinant expressed in intestinal bacteria easily forms inclusion body, and then relates to complicated change renaturation process, and whole system can produce many unfavorable factors such as a large amount of intracellular toxins.Thereby preparing interleukin 33 (rhIL-33) by traditional method has increased production cost greatly, and production technology complexity, production technique is unstable.Simultaneously, the molecular weight of current recombinant human interleukin-33 of preparing with intestinal bacteria (rhIL-33) is about 18kDa, in it is used to animal body or in human body time, very easily removed by glomerular filtration, had a strong impact on its potential physiological function and the application prospect as a drug candidate.Especially, also successfully express at present the report of the fusion rotein (rhIL33-Fc) of preparation recombinant human interleukin-33 (rhIL-33) and Fc, also for successfully not using pichia spp to express the report of preparation recombinant human interleukin-33 (rhIL-33).
To sum up, it is a kind of easy that current urgent need is wanted, active high, and output is high and have a preparation method of recombinant human interleukin-33 albumen of commercial application prospect.
Summary of the invention
The invention provides the method that one is utilized Pichia anomala expression and prepared recombinant human interleukin-33 albumen (rhIL33-Fc), it has comprised the structure of recombinant expression plasmid, screening, the optimization of engineering strain fermentation condition and the separation and purification of recombinant human interleukin-33 albumen of efficient expression strain.Realize the present invention by following steps: (1) construction of expression vector, the encoding sequence that it comprises promotor, terminator, antibiotics resistance gene, secreting signal peptide and recombinant human interleukin-33 (rhIL33-Fc); (2) electricity turns pichia spp, utilizes antibiotic concentration gradient to screen efficient cloning by expression; (3) fermentation condition of optimizing project bacterial strain; (4) separation and purification recombinant human interleukin-33 (rhIL33-Fc) albumen.
Concrete, the present invention relates to the following:
1. an expression cassette, described expression cassette from 5 ' to 3 ' comprises following element successively:
(1) pichia spp promotor;
(2) signal peptide sequence;
(3) sequence of recombinant human interleukin-33 and IgG1-Fc fusion rotein (rhIL33-Fc);
(4) transcription terminator.
2. according to the expression cassette described in 1, wherein said pichia spp promotor is the GAP promotor of pGAPZ α-A plasmid or the AOX1 promotor of pPIC9K plasmid; Described signal peptide is α-factor sequence, and its nucleotide sequence is as shown in SEQ ID No:1; The nucleotide sequence of described rhIL33-Fc is as shown in SEQ ID No:2; Described transcription terminator is the AOX1 Transcription Termination sub-element of pPIC9K plasmid.
3. an expression vector, it comprises the expression cassette described in 1-2 any one.
4. a Pichia yeast engineering, it comprises the expression vector described in 3.
5. recombinant human interleukin-33 albumen (rhIL33-Fc), its aminoacid sequence is as shown in SEQID No:3.
6. a method of efficiently preparing recombinant human interleukin-33 albumen (rhIL33-Fc), it comprises the step of expressing the Pichia yeast engineering described in 4.
7. improve a method for the expressing quantity of recombinant protein engineering strain, it is characterized in that mixed carbon source using sorbyl alcohol and other carbon source is as carbon source.
8. according to the method described in 7, the preferred glucose of wherein said other carbon source and/or glycerine.
9. according to the method described in 6, the method steps of its expressing quantity that comprises the raising recombinant protein engineering strain described in 7 or 8, wherein the blending ratio of sorbyl alcohol and other carbon source can suitably regulate, preferably 1:1,1:2 or 1; 3.
The rhIL33-Fc that method described in 10.6 or 9 is expressed.
The present invention staff finds, using recombinant human interleukin-33 (rhIL33-Fc) of the inventive method expression and preparation is a homodimer being formed by covalent disulfide bonds, dimeric molecular weight is about 120kDa (the shown molecular weight of 10%SDS-PAGE, non-theoretical molecular).Process recombinant human interleukin-33 albumen of purifying with Glycosylase F, find that molecular weight has significantly and reduces (seeing figure-2), show that prepared recombinant human interleukin-33 of the present invention (rhIL33-Fc) have N-glycosyl modified, and the glycosylation site that mass spectrum result shows as shown in Figure 3.
In addition, the present invention staff finds, uses the mixed solution of glucose solution and Sorbitol Solution USP as the carbon source of engineering strain growth, the output (seeing Fig. 6) of raising recombinant human interleukin-33 that can highly significant.The expressing quantity that significantly improves the recombinant protein engineering strain taking GAP as promotor with the mixed carbon source of glucose and sorbyl alcohol still belongs to the first time.
It is high that the inventive method is expressed output, and the post-processed of fermented liquid and purifying are convenient, have good industrialization prospect.
Detailed Description Of The Invention
Below technical scheme of the present invention is elaborated further.It should be pointed out that each embodiment of the present invention can combine as required by any way.
First aspect of the present invention provides a kind of expression cassette, and it comprises encoding sequence and the transcription terminator of pichia spp promotor, signal peptide sequence, rhIL33-Fc.Wherein said promotor is the control sequence of efficient regulation and control destination gene expression in pichia spp; Described signal peptide is secreting signal peptide, and the target protein that is about to express is secreted into the signaling molecule in the substratum of extracellular.
In a preferred embodiment, described promotor can be the GAP promotor of pGAPZ α-A plasmid or the AOX1 promotor of pPIC9K plasmid, is preferably GAP promotor.
In another preferred embodiment, can use various suitable secreting signal peptide well known to those skilled in the art, it includes but not limited to α-factor, α-amylase, glucoamylase, serum albumin secreting signal peptide, and preferred described secreting signal peptide is α-factor sequence.
In another preferred embodiment, described terminator is AOX1 transcription terminator.
In another preferred embodiment, described rhIL33-Fc is the encoding sequence of the recombinant protein of hIL-3 3 (IL33) and human IgG1 Fc, in a further preferred embodiment, IL33 is Ser112-Thr270 part, and IgG1-Fc sequence is as shown in SEQ ID No:4.
In a more preferred embodiment, the encoding sequence of recombinant human interleukin-33 used (rhIL33-Fc) albumen is without codon optimized sequence.
In the most preferred embodiment, the encoding sequence of recombinant human interleukin-33 (rhIL33-Fc) albumen used is through codon optimized sequence.It is well known to those skilled in the art that encoding sequence is optimized to the method that is beneficial to express in host cell.
Second aspect of the present invention provides a kind of expression vector, and the expression cassette that described expression vector comprises first aspect of the present invention also comprises the resistant gene for screening in described expression vector.In a preferred embodiment, described resistant gene is Zeocin resistant gene.
In a preferred embodiment, can use various pichia spp plasmid well known in the art, include but not limited to pPIC9K, pPICZ α-A/B/C, pGAPZ-A/B/C and pGAPZ α-A/B/C etc.
The 3rd aspect of the present invention provides a kind of Pichia yeast engineering of restructuring, expression cassette or expression vector that described engineering bacteria comprises the present invention the first and second aspects.In a preferred embodiment, can use various Pichi strain well known in the art, it includes but not limited to X-33, GS115 and KM71 etc.
The 4th aspect of the present invention provides the method for expressing rhIL33-Fc with the Pichia yeast engineering of third aspect of the present invention.
In a specific embodiment, be also included in the step of expressing under optimum culture condition, described optimum culture condition comprises pH, the optimization of temperature and substratum.
In a preferred embodiment, optimum culture condition comprises that medium pH is 7.5, and culture temperature is 30 degree, and the carbon source of substratum is the mixed solution of sorbyl alcohol and other carbon source.
In a preferred embodiment, other carbon source is glucose or glycerine, and the ratio of mixed solution can be adjusted.
In a most preferred embodiment, the carbon source of described substratum is for improving the expressing quantity of the recombinant protein engineering strain taking GAP as promotor.
In another specific embodiment, also comprise the step of purifying rhIL33-Fc, comprise the following steps:
(1) centrifugation thalline and fermented liquid supernatant, collects the fermentation supernatant after centrifugal, and further filtering fermentating liquid supernatant;
(2) go out recombinant human interleukin-33 albumen (rhIL33-Fc) with the affine protein A post of Fc (as MabSelect etc.) separation and purification from fermented liquid supernatant.
(3) remove residual DNA with anion-exchange chromatography post (as Q-Sepharose HP, Capto DEAE etc.), foreign protein etc.,
(4) remove multimeric protein and protein degradation fragment with molecular sieve (as S200HR etc.), obtain recombinant human interleukin-33 albumen (rhIL33-Fc) of purifying.
The rhIL33-Fc that the 5th aspect of the present invention provides the method for the 4th aspect of the present invention to express, it is not limited only to the aminoacid sequence of expressing, and in preferred embodiments, also comprises the modification to aminoacid sequence, as glycosylation modified.
Can interosculate at above-mentioned each technical characterictic of the present invention with at (as embodiment) specifically described each technical characterictic below, thereby form new or preferred technical scheme.
Brief description of the drawings
The structure schema of Fig. 1 α-factor-rhIL33-Fc/pGAPZ-A plasmid;
Fig. 2 coomassie brilliant blue staining method detects the rhIL33-Fc albumen that Glycosylase F processes;
The glycosyl modified site of N-that Fig. 3 LC-MS method detects;
Fig. 4 high efficiency engineering bacterial strain colony screening;
The cultivation pH condition of Fig. 5 optimizing project bacterial strain;
The culture temperature condition of Fig. 6 optimizing project bacterial strain;
The carbon source of Fig. 7 optimizing project strain growth.
Embodiment
Experiment material
Bacterial strain and plasmid
Pichia pastoris phaff (Pichiapastoris) strain X-33 and pGAPZ-A plasmid are purchased from Invitrogen company.
Reagent
Yeast Nitrogen Base (YNB) is purchased from BD company; Yeast Extract and Tryptone are purchased from OXOID company; D-Sorbitol (sorbyl alcohol), Biotin (vitamin H), archaeal dna polymerase, T4DNA ligase enzyme, restriction enzyme, albumen marker and PAGE configuration related reagent are purchased from the raw work in Shanghai; Plasmid extraction test kit, PCR product reclaim test kit, DNA glue reclaims test kit purchased from Axygen company; Zeocin is purchased from Invitrogen company; Glycosylase F is purchased from New EnglandBiolabs company; Goat anti-human igg-Fc HRP antibody is purchased from Thermo company; IL-33ELISA test kit is purchased from R & D company; Other chemical reagent are purchased from traditional Chinese medicines group.
Primer
Primer-1:5 ' CGGAATTCAATTCGAAACG ATGAGATTTCCTTCAATTTT3 ' (SEQ ID No:10)
Primer-2:5 ' AATTCCTGTGATGGATCTTTTCTCGAGAGATACCC3 ' (SEQ ID No:11)
Primer-3:5 ' TCTCTCGAGAAAAGATCCATCACAGGAATTTCACC3 ' (SEQ ID No:12)
Primer-4:5 ' ACAAGATTTGGGCTCAGTTTCAGAGAGCTTAAACA3 ' (SEQ ID No:13)
Primer-5:5 ' AAGCTCTCTGAAACTGAGCCCAAATCTTGTGACAA3 ' (SEQ ID No:14)
Primer-6:5 ' ATAAGAATGCGGCCGCTTATTTACCCGGAGACAGGG3 ' (SEQ ID No:15)
In following embodiment, other experimental techniques that use if no special instructions, are ordinary method.
Other material, reagent etc. used in following embodiment, if no special instructions, all can obtain from commercial channels.
Embodiment 1
The structure of α-factor-rhIL33-Fc/pGAPZ-A plasmid
1. fusion fragment α-factor-rhIL33-Fc of amplification α-factor sequence, IL-33 sequence (Ser112-Thr270) and IgG1-Fc (Glu99-Lys330)
(1) taking pPIC9K plasmid as template, carry out PCR with primer-1 and primer-2, PCR reaction conditions is: first 94 DEG C 3 minutes; Then 94 DEG C 30 seconds, 55 DEG C 40 seconds, 72 DEG C 30 seconds, totally 35 circulations; Last 72 DEG C 5 minutes.After reaction finishes, the PCR product obtaining is carried out to 1% agarose gel electrophoresis detection, obtain the band (being α-factor fragment) of an about 300bp of size, conform to expected results.Finally, reclaiming test kit with product reclaims PCR fragment.
Taking the cDNA ORF cloned plasmids (purchased from ORIGENE company) of people IL-33 as template, carry out PCR with primer-3 and primer-4, PCR reaction conditions is: first 94 DEG C 3 minutes; Then 94 DEG C 30 seconds, 55 DEG C 40 seconds, 72 DEG C 30 seconds, totally 35 circulations; Last 72 DEG C 5 minutes.After reaction finishes, the PCR product obtaining is carried out to 1% agarose gel electrophoresis detection, obtain the band (being rhIL-33 fragment) of an about 500bp of size, conform to expected results.Finally, reclaiming test kit with product reclaims PCR fragment.
(2) reclaiming product (being α-factor fragment and rhIL-33 fragment) with 2 PCR of above-mentioned (1) is template, carry out PCR (overlapping extension PCR) with primer-1 and primer-4, PCR reaction conditions is: first 94 DEG C 3 minutes; Then 94 DEG C 30 seconds, 55 DEG C 40 seconds, 72 DEG C 1 minute, totally 35 circulations; Last 72 DEG C 5 minutes.After reaction finishes, the PCR product obtaining is carried out to 1% agarose gel electrophoresis detection, obtain the band (being α-factor-rhIL33 fragment) of an about 800bp of size, conform to expected results.Finally, reclaim test kit with product PCR fragment is reclaimed, and then obtained α-factor-rhIL33 fragment.
(3) taking the cDNA cloned plasmids (purchased from ORIGENE company) of IGHG1 as template, carry out PCR with primer-5 and primer-6, PCR reaction conditions is: first 94 DEG C 3 minutes; Then 94 DEG C 30 seconds, 55 DEG C 40 seconds, 72 DEG C 1 minute, totally 35 circulations; Last 72 DEG C 5 minutes.After reaction finishes, the PCR product obtaining is carried out to 1% agarose gel electrophoresis detection, obtain the band (being IgG1-Fc fragment) of an about 700bp of size, conform to expected results.Finally, reclaiming test kit with product reclaims PCR fragment.
(4) be template by the α-factor-rhIL33 fragment obtaining in above-mentioned (2) and (3) step and IgG1-Fc fragment, carry out PCR (overlapping extension PCR) with primer-1 and primer-6, PCR reaction conditions is: first 94 DEG C 3 minutes; Then 94 DEG C 30 seconds, 55 DEG C 40 seconds, 72 DEG C 2 minutes, totally 35 circulations; Last 72 DEG C 5 minutes.After reaction finishes, the PCR product obtaining is carried out to 1% agarose gel electrophoresis detection, obtain the band (being α-factor-rhIL33-Fc fragment) of an about 1500bp of size, conform to expected results.Finally, reclaiming test kit with product reclaims PCR fragment.
2. being connected of α-factor-rhIL33-Fc fragment and pGAPZ-A plasmid
With EcoR I and Not I double digestion α-factor-rhIL33-Fc fragment and pGAPZ-A plasmid respectively, purifying reclaims α-factor-rhIL33-Fc fragment and pGAPZ-A plasmid respectively, connect with T4DNA ligase enzyme again, and transformed competence colibacillus bacillus coli DH 5 alpha, screening positive clone.Positive recombinant plasmid is entrusted the order-checking of handsome company, result shows α-factor-rhIL33-Fc fragment and pGAPZ-A plasmid exact connect ion, and α-factor-rhIL33-Fc sequence is consistent with expection, and then having obtained α-factor-rhIL33-Fc/pGAPZ-A expression plasmid, whole structure flow process is shown in figure-1.
In the above plasmid, the encoding sequence of α-factor-IL33-Fc fragment is as follows:
(ATGAGATTTCCTTCAATTTTTACTGCAGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTCCAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTACTCAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAACGGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTCTCGAGAAAAGA)<TCCATCACAGGAATTTCACCTATTACAGAGTATCTTGCTTCTCTAAGCACATACAATGATCAATCCATTACTTTTGCTTTGGAGGATGAAAGTTATGAGATATATGTTGAAGACTTGAAAAAAGATGAAAAGAAAGATAAGGTGTTACTGAGTTACTATGAGTCTCAACACCCCTCAAATGAATCAGGTGACGGTGTTGATGGTAAGATGTTAATGGTAACCCTGAGTCCTACAAAAGACTTCTGGTTGCATGCCAACAACAAGGAACACTCTGTGGAGCTCCATAAGTGTGAAAAACCACTGCCAGACCAGGCCTTCTTTGTCCTTCATAATATGCACTCCAACTGTGTTTCATTTGAATGCAAAACTGATCCTGGAGTGTTTATAGGTGTAAAGGATAATCATCTTGCTCTGATTAAAGTAGACTCTTCTGAGAATTTGTGTACTGAAAATATCTTGTTTAAGCTCTCTGAAACT>[GAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGTAAATAA](SEQ ID No:5)
Wherein, in (), be signal peptide α-factor nucleotide sequence (SEQ ID No:1); In <>, be rhIL-33 nucleotide sequence (SEQ ID No:6); In [], be IgG1-Fc nucleotide sequence (SEQ IDNo:4).α-factor aminoacid sequence (SEQ ID No:7) after translation is: MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFS NSTNNGLLFINTTIASIAAKEEGVSLEKR; The aminoacid sequence (SEQ ID No:8) of IL-33 is: SITGISPITEYLASLSTYNDQSITFALEDESYEIYVEDLKKDEKKDKVLLSYYESQ HPSNESGDGVDGKMLMVTLSPTKDFWLHANNKEHSVELHKCEKPLPDQAFFVLHNM HSNCVSFECKTDPGVFIGVKDNHLALIKVDSSENLCTENILFKLSET; The aminoacid sequence (SEQ ID No:9) of IgG1-Fc is: EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK.
Embodiment 2
α-factor-rhIL33-Fc/pGAPZ-A recombinant expression plasmid electricity transforms pichia spp
With Avr II linearizing α-factor-rhIL33-Fc/pGAPZ-A recombinant expression plasmid, ethanol precipitation reclaims.Get the plasmid after 5-10 μ g linearizing, transform cup with 0.2cm electricity and carry out electricity conversion Pichi strain X-33 (parameter setting: voltage 1500V, electric capacity 25 μ F, 200 ohm of resistance, discharge time 4-5ms), YPD plate (the 2%Tryptone that coated plate is containing Zeocin resistance, 1%YeastExtract, 2%Glucose, 1.5%Agar, 100 μ g/ml Zeocin), be positioned in 28 DEG C of biochemical cultivation cases and cultivate 3-5 days.
In the time that the mono-clonal on the YPD of Zeocin resistance plate grows to diameter 1-2mm, with the mono-clonal of aseptic toothpick picking part and be scoring to the YPD plate that contains higher Zeocin concentration, to obtain the copy number of higher IL-33 expression cassette, the concentration of Zeocin is respectively 250 μ g/ml, 500 μ g/ml, 1mg/ml and 2mg/ml.In the time that the monoclonal diameter on the YPD of above-mentioned Zeocin resistance plate is 1-2mm, picking mono-clonal carries out the expression identification (concrete grammar is shown in following examples 3 parts) of recombinant human interleukin-33 albumen.
Embodiment 3
The engineering strain of screening efficiently expressing recombinant human interleukin 33 albumen
Picking mono-clonal access the YPD substratum of 10ml from the YPD plate that contains Zeocin resistance, is placed in 28 DEG C of shaking tables, and 200rpm shakes and spends the night.Next day, in the time that the thalline OD600 in YPD culture reaches 2-6, get the culture that biomass is equivalent to 0D600=10,1500g is centrifugal and abandon supernatant.Thalline is resuspended with aseptic distilled water, and 1500g is centrifugal and abandon supernatant again.Wash thalline one time with aseptic double-distilled water again, step is the same.Finally, the thalline of centrifugal acquisition is resuspended in to BMGY substratum (2%Tryptone, 1%Yeast Extract, the 1.34%YNB of 10ml, 0.1M phosphate buffered saline buffer, 1%Glycerol, pH=6), be placed in the shaking table of 28 DEG C, 250rpm, continues to cultivate 24 hours.
Collect the culture of 24 hours, wherein part culture detects for OD600, and remaining culture is used for 12000g, 4 DEG C of centrifugal 10min, and collect supernatant and detect for follow-up ELISA.The ratio size of the concentration of recombinant human interleukin-33 that record according to ELISA and thalline OD600 value is as the foundation that judges efficient expression strain screening.That as shown in Figure 4, unit thalline output is the highest is No. 4 clones.Thereby, can filter out accurately efficient cloning by expression by the method.
Embodiment 4
The culture condition (pH and temperature) of optimizing project bacterial strain
The optimization step of pH condition is as follows: the fresh mono-clonal of picking from YPD flat board, is inoculated in incubated overnight in the YPD substratum of 25ml.Next day, get in the BMGY of different pH that the thalline that is equivalent to 10OD600 enters 10ml (pH is respectively 5,6,7 and 7.5), be placed in the shaking table of 30 degree, 250rpm, cultivates 12 hours.Subsequently, get part culture and detect for OD600, remaining culture 12000g, 4 DEG C of centrifugal 10min, and collect supernatant and detect for follow-up ELISA.The ratio size of the concentration of recombinant human interleukin-33 that record according to ELISA and thalline OD600 value is as the foundation that judges best pH.As shown in Figure 5, the fermentation pH condition that unit thalline output is the highest is pH7.5.
The optimization step of temperature condition is as follows: the fresh mono-clonal of picking from YPD flat board, is inoculated in incubated overnight in the YPD substratum of 25ml.Next day, get in the BMGY that the thalline that is equivalent to 10OD600 enters 10ml (pH7.5), be placed in the shaking table (temperature is respectively 20 degree, and 25 degree and 30 are spent) of differing temps, 250rpm, cultivates 12 hours.Subsequently, get part culture and detect for OD600, remaining culture 12000g, 4 DEG C of centrifugal 10min, and collect supernatant and detect for follow-up ELISA.The ratio size of the concentration of recombinant human interleukin-33 that record according to ELISA and thalline OD600 value is as the foundation that judges best temperature.As shown in Figure 6, the leavening temperature that unit thalline output is the highest is 30 degree.
Embodiment 5
The carbon source of optimizing project bacterial strain
Take out the work seed of a pipe engineering strain refrigerators from-80 degree, be inoculated in incubated overnight in the YPD substratum of 200ml.Next day, get that in the substratum of different carbon sources that the thalline that is equivalent to 200OD600 accesses respectively 200ml, (total medium component is 2%Tryptone, 1%YeastExtract, 1.34%YNB, 0.1M phosphate buffered saline buffer, but carbon source is respectively 2% glycerine, 2% glucose, the mixed solution of the sorbyl alcohol of 2% sorbyl alcohol and 1% glucose+1%), be placed in 250rpm, the shaking table of 30 DEG C is cultivated and after 24 hours, is received sample and detect OD600 value, and detects protein concentration with ELISA.The protein concentration size of recombinant human interleukin-33 that record according to ELISA is as the foundation that judges optimum carbon source.As shown in Figure 7, the Ro 24-7472/000-33 the highest group of protein content in supernatant is to utilize the mixed carbon source of glucose and sorbyl alcohol.
Embodiment 6
Shake flask fermentation and the purifying of recombinant human interleukin-33 albumen (rhIL33-Fc)
From-80 degree refrigerators, take out the work seed of a pipe engineering strain, be inoculated in the YPD substratum of 100ml, be placed in 30 DEG C of shaking tables, 250rpm overnight incubation.Next day, abandon after supernatant centrifugal overnight culture 1500g, with substratum (the 1%Yeast Extract of 200ml, 2%Peptone, 1.34%YNB, 0.1M phosphate buffered saline buffer, 2% glucose, 2% sorbyl alcohol, pH=7.5) resuspended thalline, is placed in 30 DEG C of shaking tables, 250rpm, continue fermentation 72 hours, wherein the mixed carbon source (1:1 mixing) of a supplementary glucose and sorbyl alcohol every 24 hours.
The purification step of recombinant human interleukin-33 albumen (rhIL33-Fc) is as follows: (1) fermented liquid, for the centrifugal 30min of 12000g at 4 DEG C, is collected fermentation supernatant.(2) with Flex Stand system and 0.45 μ m microfiltrationcartridge, the fermentation supernatant of upper step is filtered one time.(3) carry out separation and purification rhIL33-Fc albumen with the MabSelect post of XK26/20column (purchased from GE healthcare company) and MabSelect glue (purchased from GEhealthcare company) assembling.(4) with further the purify rhIL33-Fc albumen of MabSelect post wash-out of anion chromatography post, remove residual DNA and foreign protein.(5) use the super filter tube (purchased from Millipore company) of 10kDa to concentrate the rhIL33-Fc albumen of anion chromatography post wash-out.(6) remove further recombinant protein polymer and degradation fragment with the molecular sieve (purchased from GEhealthcare company) of S200HR.(7) use the super filter tube of 10kDa to concentrate the rhIL33-Fc albumen of S200HR molecular sieve purification, make the concentration of rhIL33-Fc albumen more than 1mg/ml, endotoxin content is lower than 0.1EU/ μ g albumen.
Embodiment 7
The glycosyl modified qualification in recombinant human interleukin-33
After the rhIL33-Fc albumen of final purifying is processed with Glycosylase F (purchased from NEB company), then identify with SDS-PAGE.As shown in Figure 2, the molecular weight that has added the sample of 2-ME (mercaptoethanol) is about 50～60kDa left and right, and the molecular weight that does not add the sample of 2-ME is about 120～130kDa, show that this method rhIL33-Fc albumen expressed and preparation is to link by disulfide linkage the homodimer forming.In addition, with the raw sample of going back of Glycosylase F (PNGase F) processing, its banding pattern becomes a band of homogeneous, and molecular weight becomes 45kDa left and right, approach the theoretical glycosyl modified molecular weight that do not have, this result shows, it is glycosyl modified that prepared recombinant human interleukin-33 of above method have N-.
The band of the approximately 45kDa obtaining after Glycosylase F on above-mentioned PAGE is processed send LC-MS mass spectrum center, with after the sample preparation of Trypsin enzymolysis, digestion for LC-MS mass spectrometric detection (Thermo company).If shown in 3, detected result shows 2 glycosyl modified sites of N-, is respectively Asn60 and Asn241.
Claims (10)
1. an expression cassette, described expression cassette from 5 ' to 3 ' comprises following element successively:
(1) pichia spp promotor;
(2) signal peptide sequence;
(3) sequence of recombinant human interleukin-33 and IgG1-Fc fusion rotein (rhIL33-Fc);
(4) transcription terminator.
2. expression cassette according to claim 1, wherein said pichia spp promotor is the GAP promotor of pGAPZ α-A plasmid or the AOX1 promotor of pPIC9K plasmid; Described signal peptide is α-factor sequence, and its nucleotide sequence is as shown in SEQ ID No:1; The nucleotide sequence of described rhIL33-Fc is as shown in SEQ ID No:2; Described transcription terminator is the AOX1 Transcription Termination sub-element of pPIC9K plasmid.
3. an expression vector, it comprises the expression cassette described in claim 1-2 any one.
4. a Pichia yeast engineering, it comprises expression vector claimed in claim 3.
5. recombinant human interleukin-33 albumen (rhIL33-Fc), its aminoacid sequence is as shown in SEQID No:3.
6. a method of efficiently preparing recombinant human interleukin-33 albumen (rhIL33-Fc), it comprises the step of expressing Pichia yeast engineering claimed in claim 4.
7. improve a method for the expressing quantity of recombinant protein engineering strain, it is characterized in that mixed carbon source using sorbyl alcohol and other carbon source is as carbon source.
8. method according to claim 7, the preferred glucose of wherein said other carbon source and/or glycerine.
9. method according to claim 6, the method steps of its expressing quantity that comprises the raising recombinant protein engineering strain described in claim 7 or 8, wherein the blending ratio of sorbyl alcohol and other carbon source can suitably regulate, and is preferably 1:1,1:2 or 1; 3.
10. the rhIL33-Fc that the method described in claim 6 or 9 is expressed.
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