gap和aox1启动子的联合运用加强表达蛋白

Enzyme and Microbial Technology33(2003)453–459Combined use of GAP and AOX1promoter to enhance the expression of human granulocyte-macrophage colony-stimulatingfactor in Pichia pastorisJ.M.Wu a,J.C.Lin a,L.L.Chieng a,C.K.Lee a,∗,T.A.Hsu b,ca Department of Chemical Engineering,National Taiwan University of Science and Technology,Taipei106,Taiwanb Division of Biotechnology and Pharmaceutical Research,National Health Research Institutes,Taipei115,Taiwanc Department of Chemical Engineering,National Tsing Hua University,Hsin-Chu300,TaiwanReceived17January2003;accepted8April2003AbstractGlyceraldehydes-3-phosphate dehydrogenase(GAP)promoter provides a strong constitutive expression of heterologous proteins at a level comparable that seen with the methanol-induced alcohol oxidase(AOX1)promoter in Pichia pastoris.In the present study,strain GS115/G was created to constitutively express secreted human granulocyte-macrophage colony-stimulating factor(hGM-CSF)using GAP-based expression system.With addition of1%casamino acids,the cultivation of GS115/G resulted in ca.20%higher cells concentration and ca. 30%higher level secreted protein concentration in YPD medium.The secreted proteins consisted of an unglycosylated glycosylated recombinant hGM-CSF with different extent of glycosylation.Instead of using GAP promoter alone in strain GS115/G,the strain GS115/GA that could constitutively and inducibly express recombinant hGM-CSF was generated by transforming host strain GS115/G with AOX1 promoter-controlled hGM-CSF expression cassette.In the presence of methanol,the strain GS115/GA expressed recombinant hGM-CSF at a level about two-fold higher than that constitutively expressed by GS115/G.©2003Elsevier Inc.All rights reserved.Keywords:hGM-CSF;P.pastoris;GAP promoter;AOX1promoter;Glycoprotein1.IntroductionThe methylotrophic yeast,Pichia pastoris has proven tobe an effective host for the production of both secreted andintracellular heterologous proteins[1].The high level ex-pression of heterologous proteins can be achieved using astrong,tightly regulated,and methanol-induced alcohol oxi-dase(AOX1)promoter[2].Unlike the expression from plas-mid,the heterologous genes of interest are stably integratedinto the Pastoris genome via homologous recombination[3]that eliminates the genetic instability due to segregationalloss.Furthermore,P.pastoris is capable of growing to veryhigh cell density minimal These advantagesmake P.pastoris a very attractive host for recombinant pro-teins production in a large-scale operation.Regulation and induction of heterologous gene expressionmethanol have been shown to be simple,easy to scale-up,and cost-effective for large-scale fermentations[4].How-ever,accurate regulation of the methanol concentration in P.∗Corresponding author.Tel.:+886-2-2737-6629;fax:+886-2-2737-6644.E-mail address:cklee@.tw(C.K.Lee).pastoris cultures is necessary because of the high concen-trations of methanol are toxic to P.pastoris due to the accu-mulation of formaldehyde and hydrogen peroxide insidecells[5,6].Glyceraldehydes-3-phosphate dehydrogenase(GAP)promoter has recently been developed for P.pastorisas an alternative strong constitutive promoter[7].In GAPpromoter expression system,the cloned heterologous proteinwill be expressed along with cell growth if the protein is nottoxic to the cell.Therefore,it can easily adapt to a contin-uous culture that theoretically allows indefinite productionof recombinant proteins.Human granulocyte-macrophagecolony-stimulating factor(hGM-CSF)is required for theproduction of granulocytes and macrophages from normalbone marrow and appears regulate the activity of mature,differentiated granulocytes and macrophages[8].Its genehas been cloned and expressed in Escherichia coli[9]andSaccharomyces cerevisiae[10,11].The best of our knowl-edge,its expression in P.pastoris has never been reported.In this work,the expression of secreted hGM-CSF in P.pastoris under the control of GAP promoter was studied.Most of the reported heterologous proteins expression in P.pastoris is employed using either AOX1or GAP expressionsystem alone[1].The combined use of AOX1promoter and 0141-0229/$–see front matter©2003Elsevier Inc.All rights reserved.doi:10.1016/S0141-0229(03)00147-9454J.M.Wu et al./Enzyme and Microbial Technology 33(2003)453–459GAP promoter in P .pastoris has been mentioned in the study of N -glycan engineering of glycoproteins [12].The GAP promoter-controlled enzyme is expressed to modify the AOX1promoter-controlled target protein with desired glyco-pattern.The combined use of these two promoters has also been employed to sequentially express and separately recover two different heterologous proteins from a single P .pastoris cultivation [13].In this paper,in addition to the GAP promoter expression the expression cassette under the control of AOX1promoter was also integrated into the chromosome of P .pastoris to generate a strain that could produce recombinant hGM-CSF constitutively and inducibly.The enhanced production of hGM-CSF demon-strated another advantage of combined use of GAP and AOX1promoter expression system in P .pastoris .2.Materials and methods 2.1.Strains and vector constructionThe cDNA of human colony-stimulating factor (hGM-CSF)was kindly provided by Dr.Fig.1.P .pastoris vectors for hGM-CSF expression:(a)pJM2was created by placing hGM-CSF gene under control of GAP promoter.Host strain GS115was transformed with pJM2to generate GS115/G for constitutive expression of hGM-CSF;(b)pJM1was created by placing hGM-CSF gene under control of AOX1promoter.Host strain GS115/G was transformed with pJM1to generate GS115/GA for constitutive and inducible expression of hGM-CSF.Mi-Hua Tao of IMBS,Academia Sinica.A construction fragment was generated by PCR on the cDNA of hGM-CSF using primers with the following sequences:forward 5 -AAC CGG AAT TCG CAC CCG CCC GCT CGC CCA GCC CCA GC-3 ,reverse 5 -GGC CGG AAT TCT TAG TGG TGG TGG TGG TGG TGC TCC TGG ACT GGC TCC CAG CAG TCA AAG G-3 .The resulting product contains the open reading frame of hGM-CSF fused with a six histidine tag at C-terminal,and Eco RI sites at both 5 and 3 ends.The inducible and constitutive recombinant hGM-CSF expression vectors constructed for this study are shown in Fig.1.PCR products were inserted into pPIC9K and pGAPZ ␣A vectors (Invitrogen,San Diego,CA)at Eco RI cutting site to form pJM1and pJM2vectors,respectively.Both of the plasmids carry the S.cerevisiae ␣-mating factor secretion signal downstream of the pro-moters.pJM2vector possesses the selectable marker Zeocin,which is bifunctional in both Pichia and E.coli .The pJM1vector possesses the bacterial kanamycin resistance gene which confers resistance to the related eukaryotic an-tibiotic G418necessary for selection of transformants.All the cloning steps were performed in E.coli TOP 10.P .pas-toris strain GS115(his 4)(Invitrogen,San Diego,CA)wasJ.M.Wu et al./Enzyme and Microbial Technology33(2003)453–459455used as a host strain to express and secrete the recombinant hGM-CSF.For constitutive expression of hGM-CSF,host strain GS115was transformed with pJM2to generate a con-stitutive expression strain GS115/G.The strain GS115/GA that can express hGM-CSF constitutively and inducibly was prepared by transforming GS115/G with pJM1.2.2.Pichia transformationThe hGM-CSF expression plasmids pJM1and pJM2 were digested either with Sal I Sac I and either with Bsp HI or Avr II,respectively.P.pastoris cells were made compe-tent and transformed by Electro-competent GS115and GS115/G cells(80␮l)were mixed with10␮g of appropriately linearized pJM1and pJM2plasmids in a 0.2-cm electroporation cuvettes and pulsed for∼10ms with afield strength of∼7500V/cm using a Gene (Bio-Rad Lab,Hercules,CA).Transformants GS115/G har-boring the GAP promoter-controlled hGM-CSF expression cassette were selected on Zeocin(0.1mg/ml)containing YPDS plates(1%yeast extract,2%peptone,2%glucose and 1M The generation of strain GS115/GA which can express hGM-CSF constitutively and inducibly was carried out by transforming GS115/G with methanol-inducible hGM-CSF expression cassette.The transformants were selected on minimal dextrose(MD)plates(1.34%yeast nitrogen base without amino acids,400␮g/l d-biotin,2% glucose)and then on G418(0.25mg/ml)containing YPD plates(1%yeast extract,2%peptone,2%glucose).2.3.Selection of multicopy clonesPutative clones of GS115/G harboring multiple copies of the constitutive hGM-CSF expression cassette were selected on YPD plates containing0.5,1.0,1.5and2.0mg/ml of Zeocin.Clones of GS115/GA were selected on YPD plates containing0.5,1.0,and2.0mg/ml of G418.After4days of incubation at30◦C,clones were evaluated for their ability to grow in presence of increasing concentration of the antibi-otics.Clones that survived the highest antibiotics concen-trations were cultured in test tube to screen their hGM-CSF expression level by Western blot using anti-4xHis antibody (Qiagen)as a probe.2.4.SDS–PAGE and Western blotrecombinant proteins secreted in culture medium were analyzed by SDS–PAGE(15%)and stained with Coomassie Blue.The protein concentrations were deter-mined by Bradford microassay(Bio-Rad).The proteins separated by SDS–PAGE,if necessary,were transferred onto PVDF membrane(Hybond-P,Amersham Pharmacia Biotech,Uppsala,Sweden)by electroblotting(Semi-dry Trans-Blot system,Bio-Rad,Hercules,CA).After blotting, the hGM-CSF on the membrane was detected by mouse anti-4xHis antibody(Qiagen)and followed by anti-mouse antibody conjugated with alkaline phosphatase(Sigma Chemical,St.Louis).2.5.Time course experimentsFor both the GS115/G and GS115/GA clone,100ml cul-tures were grown in500ml baffledflasks in YPD medium or with additional1%casamino acids at30◦C with shaking (250rpm).Methanol was added to every culture after glu-cose was completely consumed and at every24h thereafter to a concentration of0.5%(v/v).The glucose concentration was measured by using glucose analyzer(YSI2700,Yellow Springs,OH).protein concentration of a cultured strain was determined by measuring protein concentration of the supernatant using the YPD medium as a control. 2.6.Recombinant hGM-CSF recovery and its deglycosylationChelating Sepharose fastflow resin(Amersham Pharma-cia Biotech)was employed to recover the6xHis-tagged recombinant hGM-CSF from culture medium.Culture medium of20ml was recycled through the2ml nickel chelated resin at aflow rate of1.6ml/min forfive times. After washing with starting buffer(20mM Na2HPO4,0.5M NaCl,10mM imidazole)for5min,the bound protein was desorbed from the resin by elution buffer(20mM Na2HPO4, 0.5M NaCl,500mM imidazole).The desorbed protein solution was collected at one ml per PNGase F(Peptide:N-glycosidase F,Roche Diagnostics GmbH, Mannheim,Germany)was employed to cleave the N-linked oligosaccharide the recovered hGM-CSF.The deg-lycosylation was carried out by mixing40␮l hGM-CSF, 0.5␮l enzyme,and20␮l reaction buffer(100mM pH7.8 phosphate buffer,25mM EDTA,0.2%(w/v)SDS,and 1%(v/v)2-mercaptoethanol)at37◦C for1h.3.Results and discussion3.1.Generation of constitutive(GAP)and constitutive (GAP)-inducible(AOX1)combined Pichia clonesHGM-CSF vectors shown in Fig.1were linearized with restriction enzymes prior to transformation of P.pastoris host strain GS115.resultant constitutive expression clones GS115/G were obtained by transforming GS115 with Avr II linearized vector pJM2.Since the ability of the transformants to tolerate increasing zeocin concentrations correlates with enhanced expression cassette copy number, the clones that grow on YPD plates supplemented with the highest zeocin concentration(1.5were selected as high-copy number transformants.One of the high-copy number clones was employed for the constitutive production of hGM-CSF.The clones GS115/GA containing both GAP and AOX1promoters that controls hGM-CSF expression456J.M.Wu et al./Enzyme and Microbial Technology 33(2003)453–459cassettes were obtained by transforming the constitutive production clone GS115/G with Sal I linearized inducible expression vector pJM1.Since the pJM1vector contains a selection marker (kanamycin resistance gene)different from that in pJM2(Zeocin resistance gene),the clones GS115/GA grow on YPD plates supplemented with the highest an-tibiotic G418concentration (2mg/ml)were considered as high-copy number clones and employed for the combined constitutive and inducible production of hGM-CSF.A 6xHis tag was designed to fuse C-terminal of hGM-CSF,fore,the expression of hGM-CSF in the selected clones of GS115/G and GS115/GA was confirmed by Western blot analysis of their culture media using anti-His antibody.3.2.Constitutive expression of hGM-CSFThe strain GS115/G with high-copy number of hGM-CSF expression cassette was cultivated in YPD mediumfor Fig.2.Effect of 1%casamino acids (CA)on cell growth and constitutive expression of hGM-CSF in P .pastoris GS115/G.(a)Growth curve of GS115/G in YPD (open symbol)and YPD with 1%CA addition (closed symbol),methanol was added to 0.5%(v/v)started at 24h and every 24h thereafter.( ,᭡)glucose;(᭺,᭹)optical density;(ᮀ,᭿)secreted protein concentration.(b)SDS–PAGE analysis of YPD and YPD +CA culture medium of 1–4days cultivation.the constitutive expression and secretion of hGM-CSF.It has been reported that by including 1%casamino acids in the culture medium can minimize the extracellular pro-tease activity and increase the yield of expressed protein [14].Therefore,1%casamino acids was included in YPD medium to prevent the constitutively expressed and secreted hGM-CSF from degradation throughout the entire time-span of cultivation.For comparison,a parallel culture using YPD medium only was run for the same As shown in Fig.2a ,GS115/G reveals a slightly high specific growth rate in presence of casamino acids.In both media,the glucose was completely consumed after 1day of cultivation but the cell kept the same growth rate till second day.To maintain cells growth,methanol as a carbon source was added to both cultures to 0.5%(v/v)at 24h and every 24h there-after.Since the constitutive GAP promoter was employed to control the expression of secreted hGM-CSF,as expected,the secreted protein concentration increased with the cellJ.M.Wu et al./Enzyme and Microbial Technology 33(2003)453–459457growth.With 1%casamino acids addition,cell concen-tration as well as secreted protein concentration were about 20and respectively,higher than that without casamino acids.Evidently,the casamino acids provided additional nutrition for the cells to grow into a higher concentration,as a consequence,expressed and secreted a higher level of recombinant hGM-CSF.As shown in the SDS–PAGE analy-sis (Fig.2b ),a minor band corresponding to ∼16kDa and a dominant smear of molecular mass >27kDa were observed in the cultures after 2days of cultivation.Concentrations of the detected bands of the YPD +CA medium were higher than that of YPD medium and increased with culture time accordingly.There were faint bands appeared between ∼16and ∼20kDa and turned stronger after 4days of culti-vation in YPD +CA medium.The observed concentration increase in the detected bands was in accordance with the trend of secreted protein concentration (Fig.2a ).The higherFig.4.Effect of the addition of inducible expression cassette to constitutive expression strain on the enhancement of hGM-CSF production:(a)growth curve of strain GS115/G (open symbol)and GS115/GA (closed symbol)cultivated in YPD +CA medium.Methanol was added to 0.5%(v/v)started at 24h and every 24h thereafter.( ,᭡)glucose;(᭺,᭹)optical density;(ᮀ,᭿)secreted protein concentration;(b)SDS–PAGE analysis of GS115/GA (lanes 2 –5 ),GS115/G (lanes 2–5),and GS115(lane C)culture medium of 2–5days cultivation.Fig. 3.SDS–PAGE and Western blot analysis of secreted hGM-CSF expressed by GS115/G in YPD +CA culture medium:(1)before and (2)after deglycosylation treatment with PNGase F.458J.M.Wu et al./Enzyme and Microbial Technology33(2003)453–459recombinant hGM-CSF concentration in YPD+CA mediumwas probably resulted from the higher cell concentrationand reduced protease activity toward against hGM-CSF.YPD+CA medium was,employed for the rest ofrecombinant hGM-CSF expression studies.Since there are two potential N-linked glycosylationsites on hGM-CSF sequence,most of the observed bandson SDS–PAGE probably are considered as recombinanthGM-CSF with different glycosylation extent.Only the ∼16kDa protein is considered as the unglycosylated re-combinant hGM-CSF because its molecular weight matcheswith that calculated from nucleotide sequence kDa).The existence of unglycosylated recombinant hGM-CSFsuggests that due to the strong GAP promoter theflow ofnewly synthesized␣-MF-hGM-CSF-6xHis fusion proteinthrough the secretory pathway might be too fast to allowsufficient or complete glycosylation.In contrast,the signalpeptide processing protease KEX2on the secretory pathwayhas a cleavage rate fast enough to release the␣-MF signalsequence from the unglycosylated fusion protein.To con-firm the secreted recombinant hGM-CSF was glycosylated,the proteins recovered from culture medium was deglyco-sylated using PNGase F as demonstrated in SDS–PAGEand Western blotting analysis(Fig.3).All the secretedmaterials with molecular weight other than∼16kDa wereconversed into a band with a molecular weight slightlyhigher than that of unglycosylated hGM-CSF after degly-cosylation of N-linked saccharides.Several investigators[11,15,16]have reported that the signal sequence␣-MF ofsome secreted fusion products are not able to be cleavedby KEX2when the secreted products are over-expressed.The molecular weight of␣-MF is about10kDa,there-fore,the observed new protein band after PNGase Ftreatment to remove the N-linked oligosaccharides shouldnot be unglycosylated␣-MF-hGM-CSF-6xHis fusionprotein26kDa).Since O-linked glycosylation of re-combinant proteins have been shown to be present in P.pastoris and the O-linked saccharides are generally shortas compared with N-linked saccharides[17],the unex-pected protein was probably O-glycosylated.As a conse-quence,it possessed a molecular weight slightly higherthan that of unglycosylated recombinant hGM-CSF.UnlikeSDS–PAGE,the unglycosylated and deglycosylated recom-binant hGM-CSF shows a stronger intensity than that ofglycosylated in Western blot(Fig.3).Thus,it indicates thatthe interaction between the6xHis tagged on hGM-CSF andanti-His antibody would be interfered with the N-linkedoligosaccharides of the glycosylated recombinant hGM-CSF.3.3.Constitutive-inducible combined expression ofhGM-CSFThe recombinant hGM-CSF have been constitutively ex-pressed and secreted by strain GS115/G to a level of90mg/las shown in Fig.2a.In order to increase its expression level further,promoter-controlled hGM-CSF expression cassette was integrated into the chromosome of GS115/G to generate strain GS115/GA.A higher hGM-CSF expres-sion level can be expected for GS115/GA when the AOX1 promoter is induced to operate by adding methanol after the exhaustion of glucose.cell growth and level of se-creted protein expression of both GS115/G and GS115/GA strain are compared in Fig.4a.For the strain GS115/G, the secreted protein concentration increased with the cell growth and leveled-off at90mg/l after the cells reached its stationary phase.As expected,the secreted protein of GS115/G is a growth-related product because only the con-stitutive GAP promoter is employed to control the expres-sion of hGM-CSF.On the other the strain GS115/GA resulted in a cell concentration about10%lower than that of GS115/G at The secreted pro-tein concentration,however,was about two-fold(180mg/l) higher than that of GS115/G.The lower cell concentra-tion and higher secreted protein concentration suggests that,instead of being used for growth,part of resources of GS115/GA were diverted to express the recombinant hGM-CSF when methanol was added to turn on the AOX1 Fig.4b shows that SDS–PAGE analysis of se-creted proteins produced by strain GS115/G and GS115/GA with respect to culture time.The characteristic unglycosylated band(∼16kDa)and highly glycosylated smear(30–42.7kDa)of recombinant hGM-CSF were not detected in culture medium of control strain How-ever,the intensities of the two characteristic bands produced by GS115/GA were much stronger than that produced by GS115/G.This again demonstrated that the expression level of recombinant hGM-CSF can be greatly enhanced by combined use of GAP and AOX1promoter in P.pastoris strain GS115.Since6xHis peptide has been designedto Fig. 5.SDS–PAGE analysis of secreted proteins recovered from GS115/GA and GS115/G culture medium after6days of cultivation using Ni-chelating Sepharose column.Most of proteins appeared at fractions 3–5while eluting with0.5M imidazole.J.M.Wu et al./Enzyme and Microbial Technology33(2003)453–459459fuse to C-terminal of hGM-CSF,immobilized metal affinity chromatography(IMAC)was applied to recover the recom-binant hGM-CSF from culture medium.Based on the pro-tein concentration,about80%of the secreted proteins were recovered from the cultured medium.The SDS–PAGE anal-ysis of the recovered secreted proteins of both GS115/GA and GS115/G strains is shown in Fig.5.As expected,the amount of proteins recovered from GS115/GA is much higher than that from GS115/G.The characteristic ungly-cosylated and highly glycosylated recombinant hGM-CSF bands were also observed.This indicates that oligosaccha-rides on the glycosylated recombinant hGM-CSF will not prevent its6xHis tag from binding with the immobilized nickel ion.4.ConclusionThe addition of1%casamino acids was beneficial for the cell growth and constitutive expression of recombinant hGM-CSF of P.pastoris strain GS115/G cultivated in YPD medium.The cell concentration and expressed secreted protein concentration of YPD+CA culture were about20 and30%,respectively higher than that of YPD culture. The secreted recombinant hGM-CSF showed two major characteristic molecular weights distribution.One is ung-lycosylated recombinant hGM-CSF of molecular weight about16kDa.The other is recombinant hGM-CSF with dif-ferent glycosylation extent of molecular weight30–43kDa. Recombinant hGM-CSF could be constitutively and in-ducibly expressed by strain GS115/GA which was created by transforming GS115/G with AOX1-controlled hGM-CSF expression vector.In the presence of methanol induction, the strain GS115/GA grew to a cell concentration10% lower and produced secreted hGM-CSF with a level about two-fold higher than that of GS115/G.The oligosaccha-rides on the glycosylated recombinant hGM-CSF would not prevent anti-His antibody and immobilized nickel ion from binding with the6xHis tag fused on the C-terminal of hGM-CSF.AcknowledgmentsWe thank Dr.Mi-Hua Tao(IMBS,Academia Sinica, Taiwan)for the plasmids carrying the cDNA sequence of hGM-CSF.The work was founded by National Science Council of Taiwan(NSC-90-2214-E-011-005).References[1]Cereghino JL,Cregg JM.Heterologous protein expression inthe methylotrophic yeast Pichia pastoris.FEMS Microb Rev 2000;24:45–66.[2]Tschopp JF,Brust PF,Cregg JM,Stillman CA,Gingeras TR.Expression of the lac Z gene from two methanol regulated promoters in Pichia pastoris.Nucleic Acids Res1987;15:3859–76.[3]Cregg JM,Madden KR,Barringer KJ,Thill GP,Stillman CA.Functional characterization of the two alcohol oxidase genes from the yeast Pichia pastoris.Mol Cell Biol1989;9:1316–23.[4]Cregg JM,Vedvick TS,Raschke WC.Recent advances in theexpression of foreign genes in Pichia pastoris.Bio/technology 1993;11:905–10.[5]Couderc R,Baratti J.Oxidation of methanol by the yeast,Pichiapastoris.Purification and properties of the alcohol oxidase.Agric Biol Chem1980;44:2279–89.[6]Murray WD,Duff SJB,Lanthier PH.Induction and stability ofalcohol oxidase in the methylotrophic yeast Pichia pastoris.Appl Microbiol Biotechnol1989;32:95–100.[7]Waterham HR,Digan ME,Koutz PJ,Lair SV,Cregg JM.Isolationof the Pichia pastoris glyceraldehydes-3-phosphate dehydrogenase gene and regulation and use of its promoter.Gene1997;186:37–44.[8]Metcalf D.The molecular biology and functions of the granulocyte-macrophage colony-stimulating factors.Blood1986;67:257–67. 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