Andersen, Mikael Rørdam

Kaas, Christian Schrøder

Kildegaard, Helene Faustrup

Kristensen, Claus

Lund, Anne Mathilde

The Chinese hamster ovary (CHO) cell-line is the predominant mammalian industrial cell line being used to produce recombinant therapeutic proteins. Although CHO cells have been used for more than 25 years, the genome sequence was first published in 2011. So far there have been limited studies of the cell biology of the CHO cell and the potential of cell line engineering. To elucidate the poorly understood cellular processes that control and limit recombinant protein production and secretion, a system-wide study was initiated to identify possible engineering targets relevant for therapeutic protein production

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Downloaded from orbit.dtu.dk on: Dec 19, 2017RNA-seq based expression analysis of the CHO cell protein secretion pathwayLund, Anne Mathilde; Kaas, Christian Schrøder; Kildegaard, Helene Faustrup; Kristensen, Claus;Andersen, Mikael RørdamPublication date:2013Document VersionPeer reviewed versionLink back to DTU OrbitCitation (APA):Lund, A. M., Kaas, C. S., Kildegaard, H. F., Kristensen, C., & Andersen, M. R. (2013). RNA-seq basedexpression analysis of the CHO cell protein secretion pathway. Poster session presented at 3rd Annual NextGeneration Sequencing Asia Congress, Singapore, Singapore. Strategy of in silico resonstruction of CHO cell pathwaysObjectives Design and methods for RNA-Seq samplesPerspectivesReconstructed UPR pathway and RNA-Seq data analysisPP2A/Ppp2r5aERdj3QSOX1/Qsox1JNK/Mapk8PP2A/Ppp2r2aJEM1/Blzf1OSTcomplex/Stt3aUGT1/Uggt1Traf2BaxPERKHrd1/Syvn1NRF2CNX/CANXQSOX2/Qsox2Gcn2/Eif2ak4IRE1α/Ern1CrebrfTraf6Gsk3bWfs1Edem3HSP72/Hspa2IkbkeOTU1/Yod1TORC2/CRTC2Ube4bbeta−actinPP2A/Ppp2r4Atf6bGlcIIα/GanabPP2A/Ppp2r1bMKK7/Map2k7NRF1PTP−1B/Ptpn1PP2A/Ppp2r2cCasp9Creb3BtkOsp94/Hspa4lDerl3IkbkbS2PNf−yaAIP/Dab2ipCalpain1/Capn1PP2A/Ppp2r1aUBXD8/Faf2Bcl2Hsp70A1/Hspa1aCyclinD1/Ccnd1Bak1ERdj2/Dnajc11UBC6/Ube2j2PrkrirIKKA/ChukPP2A/Ppp2r5eTBPPP2A/Ppp2r2dTcp1Atf3PPI/FKBP65/Fkbp10OASISGlcI/MogsTRIB3Fbxo6Hsp110/Hsph1Hsp60/Hspd1Nf−ycGapdhClimpp63/Ckap4Irs1NFKB1HSP47/Serpinh1Casp3PPI/FKBP23/Fkbp7Myd88YY1PP2A/Ppp2r5cPP2A/Ppp2r3dFoxo1Cxcl12JAMP1/JkampERdj1/Dnajc1ERp19/Txndc12Ost4/Ost4HERP2/Herpud2Bbf2h7OSTcomplex/Dad1Fmo1UGT2/Uggt2ASK1/Map3k5AmfrUBX2/Ubxn4eIF2α/Eif2s1RACK1/Gnb2l1Nf−ybP5/Cabp2OGT/OgtCalmeginCREBH/Creb3l3BLIMP1/Prdm1Egasyn/Ces1eTisp40/Creb3l4NOX2/CybbPP1C/Ppp1r1cHsp90aa1Serp1PP2A/Ppp2cbPP2A/Ppp2caGlcIIβ/PrkcshAtf4CHOP/Ddit3Calpain2/Capn2GADD34/Ppp1r15aPdiltPP2A/Ppp2r2bTirapPpp5cEro1lbHERP1/Herpud1CRT/CALRGRP94/Hsp90b1p58^IPK/Erdj6Ppib/CyclophilinBPDI/ERp57/Pdia3EDEM1Sdf2l1PDI/Erp44BiP/Hspa5GRP170/Hyou1PDI/ERp72/Pdia4EDEM2PPI/Fkbp2/FKBP13S1PCaspase12/Casp12Tapasin/TapbpBdnfDerl2ERO1−L../Ero1lMalectin/MlecOSTcomplex/Stt3bPDI/Erp29PDI/ERp59/P4hbXbp1PRKR/Eif2ak2Bag1VAMP−B/VapbJNK/Mapk9SEC63/Sec63OST48/DdostPDI/Pdia5JNK/Mapk10VimpOSTcomplex/Tusc3OSTcomplex/Rpn1BI−1/Tmbim6NEF/BAP/Sil10.0 0.2 0.4 0.6 0.8 1.0HeightChaperone/Foldease Apoptosis related None or very low expression Refrence gene References1. B.G. Hansen et al., Versatile enzyme expression and characterization system for Aspergillus nidulans, with the Penicillium brevicompactum polyketide synthase gene from the mycophenolic acid gene cluster as a test case, Appl Environ Microbiol. (2011) 77(9):3044-51. 2.  H.H. Nour-Eldin et al., USER cloning and USER fusion: the ideal cloning techniques for small and big laboratories, Methods Mol. Biol. (2010) 643:185-200.3. S.S. Tate et al., Secreted alkaline phosphatase: an internal standard for expression of injected mRNAs in the Xenopus oocyte, FASEB J. (1990) 4(2): 227-231. RNA-seq based expression analysis of the CHO cell protein secretion pathwayAnne Mathilde Lund1, *, Christian Schrøder Kaas2, Helene Faustrup Kildegaard3, Claus Kristensen2, Mikael Rørdam Andersen1 (1) Center for Microbial Biotechnology, Department of Systems Biology, Technical University of Denmark, Kgs. Lyngby, Denmark; (2) Current address: Novo Nordisk, Maaloev, Denmark; (3) Current address: Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hoersholm, Denmark(*) amalu@bio.dtu.dkList of mouse UniProt ID’s CHO-K1 genome (RefSeq) BLASTp (aa <-> aa) List of protein id accession # (GI) bad/no hits + isoproteins tBLASTn Mouse ID <-> DNA (±3 RF)  (aa <-> DNA) CHO-K1 genome in contigs Proteome* (GI <-> GI#) A list of predicted proteins of the secretion pathway in CHO cells RNA-seq transcriptome Literature study of the protein secretion pathway The caperones CNX, GluII-a and UGT falls in clusters dieren from ohter chaperones. This could indicate the close cornection between protein fold-ing, ER stress response og apoptosis.Many of the chaperones commonly related to protein foldin seems to be higly expressed in all cell lines, but a variaion between pro-tein produces vs non-produces,  exponential growth vs stationary phase and stressed cells. E.g. the gene encoding CHOP protein is increased in expressed under stress and in stationay phase, which also have shown positive protein production by knock-down.CHO−K1_none_expCHO−K1_IgG_expCHO−K1_IgG_0%_expCHO−K1_none_staCHO−K1_IgG_staCHO−K1_IgG_0%_staCHO−K1_IgG_NaBu_1CHO−K1_IgG_NaBu_2CHO−DG44_FVIII−high_F435−1CHO−DG44_FVIII−high_F435−3CHO−DG44_FVIII−high_1C9−1CHO−DG44_FVIII−high_1C9−2CHO−DG44_FVIII−high_1C9−3CHO−DG44_FVIII−med_K19CHO−DG44_FVIII−med_K16CHO−DG44_FVIII−med_K61−1CHO−DG44_FVIII−med_K61−2CHO−DG44_FVIII−med_K61−3CHO−DG44_FVIII−med_K28_1CHO−DG44_FVIII−med_K28_2CHO−DG44_FVIII−med_K36_1CHO−DG44_FVIII−med_K36_2CHO−DG44_FVIII−med_K36_3CHO−DG44_FVIII−med_K46CHO−DG44_FVIII−none_K52CHO−DG44_FVIII−none_K50PP2A/Ppp2r5aQSOX1/Qsox1ERdj3JNK/Mapk8PP2A/Ppp2r2aBaxPERKHrd1/Syvn1Traf2UGT1/Uggt1JEM1/Blzf1OSTcomplex/Stt3aCrebrfIRE1../Ern1Gcn2/Eif2ak4Traf6Gsk3bOTU1/Yod1IkbkeWfs1HSP72/Hspa2Edem3NRF2QSOX2/Qsox2CNX/CANXPrkrirUBC6/Ncube2/Ube2j2IKKA/ChukPP2A/Ppp2r5eTBPPP2A/Ppp2r2dTcp1Hsp70A1/Hspa1aCyclinD1/Ccnd1Bak1ERdj2/Dnajc11Bcl2UBXD8/Faf2PP2A/Ppp2r1aS2PIkbkbNf−yaCalpain1/Capn1AIP/Dab2ipBtkOsp94/Hspa4lDerl3PP2A/Ppp2r2cCasp9Creb3PTP−1B/Ptpn1TORC2/CRTC2Ube4bPP2A/Ppp2r1bMKK7/Map2k7NRF1Atf6bGlcII../GanabPP2A/Ppp2r4beta−actinGlcI/MogsFbxo6TRIB3Atf3OASISPPI/FKBP65/Fkbp10PPI/FKBP23/Fkbp7Casp3Myd88YY1PP2A/Ppp2r5cIrs1NFKB1HSP47/Serpinh1Hsp110/Hsph1Nf−ycHsp60/Hspd1Climpp63/Ckap4GapdhPP2A/Ppp2r3dCxcl12Foxo1JAMP1/JkampERdj1/Dnajc1ERp19/Txndc12HERP2/Herpud2Ost4/Ost4Bbf2h7OSTcomplex/Dad1Fmo1ASK1/Map3k5UGT2/Uggt2AmfrUBX2/Ubxn4P5/Cabp2OGT/OgteIF2../Eif2s1Nf−ybRACK1/Gnb2l1BLIMP1/Prdm1Egasyn/Ces1eTisp40/Creb3l4CalmeginCREBH/Creb3l3PP1C/Ppp1r1cNOX2/CybbGlcII../PrkcshCHOP/Ddit3Atf4PP2A/Ppp2caPP2A/Ppp2cbSerp1Hsp90aa1Calmegin/PdiltPP2A/Ppp2r2bTirapPpp5cGADD34/CreP/Ppp1r15aCalpain2/Capn2S1PCaspase12/Casp12Tapasin/TapbpBdnfDerl2ERO1−L../Ero1lOSTcomplex/Stt3bMalectin/MlecOSTcomplex/Tusc3OSTcomplex/Rpn1NEF/BAP/Sil1BI−1/Tmbim6JNK/Mapk10VimpJNK/Mapk9VAMP−B/VapbSEC63/Sec63PDI/Pdia5OST48/DdostPRKR/Eif2ak2Bag1PDI/Erp29Xbp1PDI/ERp59/P4hbPPI/Fkbp2/FKBP13EDEM2EDEM1Sdf2l1PDI/Erp44PDI/ERp72/Pdia4ORP150/GRP170/Hyou1BiP/Hspa5Ero1lbHERP1/Herpud1p58^IPK/Erdj6Ppib/CyclophilinBPDI/ERp57/Pdia3CRT/CALRGRP94/Hsp90b10 4000 Value0300Color Key and HistogramCountPERK-BiPBiPunfolded proteinsPERKdimerize and +PPERK-PeIF2α -P+PeIF2αTranslationTranslationNRF2 Transcription of AARE elementsp58IPK(Erdj6)p52rIPKGrp78vaAFT4- P GADD34 CHOP(GADD153) Bcl2Low levelsNFkBTrancription ERS elementsBAG-1BAX/BAKDR5TRB3ERO1ATF6PP1CBiPunfolded proteinsATF6monomerization+ glycosylationATF6translocation to GolgiWFS1ATF6SP1SP2 proteolysisATF6 p50NF-Y, YY1TBPXBP1sTrancription ERS elementspro-survival factorsBiP, PDI, EDEM, IRE1, XBP1, Bcl2ATF6 p50IRE1-BiPBiPunfolded proteinsIRE1αdimerize and +PIRE1α-PsXBP1(mRNA)uXBP1(mRNA)?ligaseB1-1PP2ACRTUPR elementsERAD factorsERS elementsJNKApoptosisafter prolonged activationafter prolonged activationRANCB1-1- P+PRACK1COPIIATF3TRAF2IRE1α-TRAF2 ASK1Atk, PP5Bcl2BAX/BAKCAPN1/2CASP12JNKApoptosisMKK7AIP1ATF3 B1-1HSP72CHOP(GADD153) IKKNFκB+P+P+P+P+PThe genes there are not ex-pressed or at very low levels across all samples are com-monly related to be tissue specic in mouse.Proteins associated or linked to UPR pathway were identied by manually curate available litterature on mouse models and cells lines. Furthermore, was the know interactions and dynamics of proteins in the UPR pathway mapped. Found proteins were used to iden-tify CHO-K1 genes of the UPR pathway.Identied genes and mRNA of CHO-K1Protein known to be involved in the apoptosic pathway and have a direct interaction is also seen to cluster across the samples in the RNA-Seq data. However, apoptosis can be induced trough dierent path of the UPR pathway dependend on  the cause of stress, which  is likely the reason for some of theapop-tosis genes to clustered  more closely to chaperones and fold-ing proteinsFirst a study was initiated foridentiyng mouse proteins inthe litterature associated with the secretionpathway and the ER stress responses. Amino acid sequences were used for BLASTp against the available RefSeq CHO-K1 genome. The identied genes could be used for extracting parts of the mapped RNA-seq data. However, only mapped genesThe CHO-K1 genome was se-quencing recently and the an-notation is at present insu-cient, so more than 4 mio. reads was form each sample not possible to map.Control of mapping IGV5exp. growthCHO-K1_none CHO-K1_IgGstationaryexp. growthcontrol1 samples 1 samplesCHO-DG44_FVIIInone medium high5 samples 11 samples 2 samplesexponential growth•  RNA was extracted under dierent growth condidations and treatment from three dierent CHO cell lines•  Paired-end RNA sequencing was performed by AROS a/s on Illumina HiSeq 2000 platform with a sequencing         depth of min. 35 mio readsNaBu2 samplesstationary0%NEAA control 0%NEAA1 sample 1 sample 1 sample1 sampleResync paired-end readscmpfastq3Unique single readsCommonpaired-end readsMapping of readsTophat24Cluster analysisSpearman correlation8Dierential expressionEdgR7Normalization of counts between samplesEdgR7Filtering and trimmingPrinseq-lite2QC of readsFastQC1Workow of RNA-Seq data handling and analysisOverview of samples• Reconstruction of the complex cellular machineries of the early protein secretion  pathway  and unfolded protein responses by employing legacy knowledge of mouse• By using  RNA-seq data, a dierential gene expression analysis of the constructed  CHO secretion pathway would provide a unique possibility for identication of active  components to increase the productivity of recombinant proteins. IntroductionThe Chinese hamster ovary (CHO) cell-line is the predominant mammalian industrial cell line being used to produce recombinant thera-peutic proteins. Although CHO cells have been used for more than 25 years, the genome sequence was first published in 2011. So far there have been limited studies of the cell biology of the CHO cell and the potential of cell line engineering. To elucidate the poorly un-derstood cellular processes that control and limit recombinant protein production and secretion, a system-wide study was initiated to identify possible engineering targets relevant for therapeutic protein production. This preliminary study shows the possibilites for using RNA-seq data and cluster analysis  to identify new gene clusters based on biological gene expression behaviour that can lead to a greater biological understading of the important industrial cell CHO-K1. n be used for identifying genetic targets for improvement of protein production by overexpression transcription fac-tors og create knock-downs of  growth inhibiting. 

RNA-seq based expression analysis of the CHO cell protein secretion pathway

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RNA-seq based expression analysis of the CHO cell protein secretion pathway

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