Abstract
A combined transcriptome and proteome analysis was carried out to identify key genes and proteins differentially expressed in Chinese hamster ovary (CHO) cells producing high and low levels of dhfr-GFP fusion protein. Comparison of transcript levels was performed using a proprietary 15 K CHO cDNA microarray chip, whereas proteomic analysis was perfomed using iTRAQ quantitative protein profiling technique. Microarray analysis revealed 77 differentially expressed genes, with 53 genes upregulated and 24 genes downregulated. Proteomic analysis gave 75 and 80 proteins for the midexponential and stationary phase, respectively. Although there was a general lack of correlation between mRNA levels and quantitated protein abundance, results from both datasets concurred on groups of proteins/genes based on functional categorization. A number of genes (20%) and proteins (45 and 23%) were involved in processes related to protein biosynthesis. We also identified three genes/proteins involved in chromatin modification. Enzymes responsible for opening up chromatin, Hmgn3 and Hmgb1, were upregulated whereas enzymes that condense chromatin, histone H1.2, were downregulated. Genes and proteins that promote cell growth (Igfbp4, Ptma, S100a6, and Lgals3) were downregulated, whereas those that deter cell growth (Ccng2, Gsg2, and S100a11) were upregulated. Other main groups of genes and proteins include carbohydrate metabolism, signal transduction, and transport. Our findings show that an integrated genomic and proteomics approach can be effectively utilized to monitor transcriptional and posttranscriptional events of mammalian cells in culture.
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Chu, L. and Robinson, D. K. (2001) Industrial choices for protein production by large scale cell culture. Curr. Opin. Biotechnol. 12, 180–187.
Anderson, D. C. and Krummen, L. (2002) Recombinant protein expression for therapeutic applications. Curr. Opin. Biotechnol. 13, 117–123.
Sautter, K. and Enenkel, B. (2005) Selection of high-producing CHO cells using NPT selection marker with reduced enzyme activity. Biotechnol. Bioeng. 89, 530–538.
Brezinsky, S. C., Chiang, G. G., Szilvasi, A., et al. (2003) A simple method for enriching populations of transfected CHO cells for cells of higher specific productivity. J. Immunol. Methods 277, 141–155.
Pu, H., Cashion, L. M., Kretschmer, P. J., and Liu, Z. (1998) Rapid establishment of high-producing cell lines using dicistronic vectors with glutamine synthetase as the selection marker. Mol. Biotechnol. 10, 17–25.
Ross, P. L., Huang, Y. N., Marchese, J. N., et al. (2004) Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol. Cell Proteomics 3, 1154–1169.
Wlaschin, K. F., Nissom, P. M., Gatti, M. L., et al. (2005) EST sequencing for gene discovery in Chinese hamster ovary cells. Biotechnol. Bioeng. 91, 592–606.
Brazma, A., Hingamp, P., Quackenbush, J., et al. (2001) Minimum information about a microarray experiment (MIAME)-toward standards for microarray data. Nat. Genet. 29, 365–371.
Yang, Y. H., Dudoit, S., Luu, P., et al. (2002) Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation. Nucleic Acids Res. 30, e15.
Livak, K. J. and Schmittgen, T. D. (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25, 402–408.
Meng, Y. G., Liang, J., Wong, W. L., and Chisholm, V. (2000) Green fluorescent protein as a second selectable marker for selection of high producing clones from transfected CHO cells. Gene 242, 201–207.
Unwin, R. D., Smith, D. L., Blinco, D., et al. (2006) Quantitative proteomics reveals post-translational control as a regulatory factor in primary hematopoietic stem cells. Blood 107, 4687–4694.
Gygi, S. P., Rochon, Y., Franza, B. R., and Aebersold, R. (1999) Correlation between protein and mRNA abundance in yeast. Mol. Cell Biol. 19, 1720–1730.
Anderson, L. and Seilhamer, J., (1997) A comparison of selected mRNA and protein abundances in human liver. Electrophoresis 18, 533–537.
Grandi, P., Rybin, V., Bassler, J., et al. (2002) 90S preribosomes include the 35S pre-rRNA, the U3 snoRNP and 40S subunit processing factors but predominantly lack 60S synthesis factors. Mol. Cell. 10, 105–115.
Milkereit, P., Kuhn, H., Gas, N., and Tschochner, H. (2003) The pre-ribosomal network. Nucleic Acids Res. 31, 799–804.
Fernandez-Pol, J. A., Klos, D. J., and Hamilton, P. D. (1993) A growth factor-inducible gene encodes a novel nuclear protein with zinc finger structure. J. Biol. Chem. 268, 21,198–21,204.
Suzuki, K., Olvera, J., and Wool, I. G. (1991) Primary structure of rat ribosomal protein S2. A ribosomal protein with arginine-glycine tandem repeats and RGGF motifs that are associated with nucleolar localization and binding to ribonucleic acids. J. Biol. Chem. 266, 20,007–20,010.
Brogna, S., Sato, T. A., and Rosbash, M. (2002) Ribosome components are associated with sites of transcription. Mol. Cell 10, 93–104.
Oliver, E. R., Saunders, L., Tarle, S. A., and Glaser, T. (2004) Ribosomal protein L24 defect in belly spot and tail (Bst), a mouse minute. Development 131, 3907–3920.
Tornow, J. and Santangelo, G. M. (1994) Saccharomyces cerevisiae ribosomal protein L37 is encoded by duplicate genes that are differentially expressed. Curr. Genet. 25, 480–487.
Shin, B. K., Wang, H., Yim, A. M., et al. (2003) Global profiling of the cell surface proteome of cancer cells uncovers and abundance of proteins with chaper-one function. J. Biol. Chem. 278, 7607–7616.
Shuda, M., Kondoh, N., Imazeki, N., et al. (2003) Activation of the ATF6, XBP1 and grp78 genes in human hepatocellular carcinoma: a possible involvement of the ER stress pathway in hepatocarcinogenesis. J. Hepatol. 38, 605–614.
Ito, Y. and Bustin, M. (2002) Immunohistochemical localization of the nucleosome-binding protein HMGN3 in mouse brain. J. Histochem. Cytochem. 50, 1273–1275.
Travers, A. A. (2003) Priming the nucleosome: a role for HMGB proteins? EMBO Rep. 4, 131–136.
Bi, J. X., Shuttleworth, J., and Al-Rubeai, M. (2004) Uncoupling of cell growth and proliferation results in enhancement of productivity in p21CIP1-arrested CHO cells. Biotechnol. Bioeng. 85, 741–749.
Meents, H., Enenkel, B., Eppenberger, H. M., Werner, R. G., and Fussenegger, M. (2002) Impact of coexpression and coamplification of sICAM and antiapoptosis determinants bcl-2/bcl-x(L) on productivity, cell survival, and mitochondria number in CHO-DG44 grown in suspension and serum-free media. Biotechnol. Bioeng. 80, 706–716.
Ingham, R. J., Gish, G., and Pawson, T. (2004) The Nedd4 family of E3 ubiquitin ligases: functional diversity within a common modular architecture. Oncogene 23, 1972–1984.
Pham, N. and Rotin, D. (2001) Nedd4 regulates ubiquitination and stability of the guanine-nucleotide exchange factor CNrasGEF. J. Biol. Chem. 276, 46,995–47,003.
Vecchione, A., Marchese, A., Henry, P., Rotin, D., and Morrione, A. (2003) The Grb10/Nedd4 complex regulates ligand-induced ubiquitination and stability of the insulin-like growth factor I receptor. Mol. Cell. Biol. 23, 3363–3372.
Stapulionis, R., Kolli, S., and Deutscher, M. P. (1997) Efficient mammalian protein synthesis requires an intact F-actin system. J. Biol. Chem. 272, 24,980–24,986.
Rai, M. and Padh, H. (2001) Expression systems for production of heterologous proteins. Curr. Sci. 80, 1121–1128.
Condeelis, J. (1995) Elongation factor 1α, translation and the cytoskeleton. Trends Biochem Sci. 20, 169–170.
Conrads, K. A., Yi, M., Simpson, K. A., et al. (2005) A combined proteome and microarray investigation of inorganic phosphate-induced pre-osteoblast cells. Mol. Cell. Proteomics 4, 1284–1296.
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Nissom, P.M., Sanny, A., Kok, Y.J. et al. Transcriptome and proteome profiling to understanding the biology of high productivity CHO cells. Mol Biotechnol 34, 125–140 (2006). https://doi.org/10.1385/MB:34:2:125
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DOI: https://doi.org/10.1385/MB:34:2:125