Dectin-1 (CLEC7A) is a C-type lectin receptor that binds to β-glucans found in fungal cell walls to act as a major pattern recognition receptor (PRR). Since β-glucans epitope is not present in human cells, we are of the opinion that Dectin-1 can have therapeutic functions against fungal infections. We thus set out to produce a soluble extracellular domain of murine Dectin-1 (called sDectin-1) in sufficient titers to facilitate such studies in mouse models. Since sDectin-1 has previously been shown to be glycosylated, we chose to produce this protein using Chinese Hamster Ovary (CHO) cells, a mammalian host cell line suitable for the high-titer production of recombinant glycoproteins. To ensure a high titer production of sDectin-1 and minimize the effects of gene fragmentation, we constructed a mammalian expression vector with a PEST-destabilized dhfr amplifiable marker downstream of an attenuated IRES element, which was in turn downstream of the sDectin-1 gene and a CMV IE promoter. Stably transfected and MTX-amplified cell pools were generated using this vector, and maximum sDectin-1 titers of 246 mg/l and 598 mg/l were obtained in shake flask batch culture and bioreactor fed-batch culture respectively. The purified recombinant sDectin-1 was shown to be glycosylated. Protein functionality was also demonstrated by its ability to bind to zymosan particles and to the cell wall of Saccharomyces cerevisiae. We describe for the first time the use of an attenuated IRES-linked PEST-destabilized dhfr amplifiable marker for the production of recombinant proteins with stably amplified cell pools. With our process, we reached the highest reported titer for producing recombinant proteins smaller than 50 kDa in cell pools. sDectin-1 protein produced is glycosylated and functional. This vector design can thus be used efficiently for the high-titer production of functional recombinant proteins.
References
[1]
Ariizumi K, Shen GL, Shikano S, Xu S, Ritter R 3rd, et al. (2000) Identification of a novel, dendritic cell-associated molecule, dectin-1, by subtractive cDNA cloning. J Biol Chem 275: 20157–67.
[2]
Brown GD, Gordon S (2001) Immune recognition. A new receptor for beta-glucans. Nature 413: 36–7.
[3]
Yokota K, Takashima A, Bergstresser PR, Ariizumi K (2001) Identification of a human homologue of the dendritic cell-associated C-type lectin-1, dectin-1. Gene 272: 51–60.
[4]
Brown GD, Herre J, Williams DL, Willment JA, Marshall AS, et al. (2003) Dectin-1 mediates the biological effects of beta-glucans. J Exp Med 197: 1119–24.
[5]
Steele C, Marrero L, Swain S, Harmsen AG, Zheng M, et al. (2003) Alveolar macrophage-mediated killing of Pneumocystis carinii f. sp. muris involves molecular recognition by the Dectin-1 beta-glucan receptor. J Exp Med 198: 1677–88.
[6]
Viriyakosol S, Fierer J, Brown GD, Kirkland TN (2005) Innate immunity to the pathogenic fungus Coccidioides posadasii is dependent on Toll-like receptor 2 and Dectin-1. Infect Immun 73: 1553–60.
[7]
Steele C, Rapaka RR, Metz A, Pop SM, Williams DL, et al. (2005) The beta-glucan receptor dectin-1 recognizes specific morphologies of Aspergillus fumigatus. PLoS Pathog 1: e42.
[8]
Herre J, Marshall AS, Caron E, Edwards AD, Williams DL, et al. (2004) Dectin-1 uses novel mechanisms for yeast phagocytosis in macrophages. Blood 104: 4038–45.
[9]
Gantner BN, Simmons RM, Canavera SJ, Akira S, Underhill DM (2003) Collaborative induction of inflammatory responses by dectin-1 and Toll-like receptor 2. J Exp Med 197: 1107–17.
[10]
Underhill DM, Rossnagle E, Lowell CA, Simmons RM (2005) Dectin-1 activates Syk tyrosine kinase in a dynamic subset of macrophages for reactive oxygen production. Blood 106: 2543–50.
[11]
Rogers NC, Slack EC, Edwards AD, Nolte MA, Schulz O, et al. (2005) Syk-dependent cytokine induction by Dectin-1 reveals a novel pattern recognition pathway for C type lectins. Immunity 22: 507–17 Erratum in: Immunity. 2005. 22:773–4.
[12]
Taylor PR, Brown GD, Reid DM, Willment JA, Martinez-Pomares L, et al. (2002) The beta-glucan receptor, dectin-1, is predominantly expressed on the surface of cells of the monocyte/macrophage and neutrophil lineages. J Immunol 169: 3876–82.
[13]
Reid DM, Montoya M, Taylor PR, Borrow P, Gordon S, et al. (2004) Expression of the beta-glucan receptor, Dectin-1, on murine leukocytes in situ correlates with its function in pathogen recognition and reveals potential roles in leukocyte interactions. J Leukoc Biol 76: 86–94.
[14]
Taylor PR, Tsoni SV, Willment JA, Dennehy KM, Rosas M, et al. (2007) Dectin-1 is required for beta-glucan recognition and control of fungal infection. Nat Immunol 8: 31–8.
[15]
Saijo S, Fujikado N, Furuta T, Chung SH, Kotaki H, et al. (2007) Dectin-1 is required for host defense against Pneumocystis carinii but not against Candida albicans. Nat Immunol 8: 39–46.
[16]
Yoshitomi H, Sakaguchi N, Kobayashi K, Brown GD, Tagami T, et al. (2005) A role for fungal {beta}-glucans and their receptor Dectin-1 in the induction of autoimmune arthritis in genetically susceptible mice. J Exp Med 201: 949–60.
[17]
Plantinga TS, Fransen J, Takahashi N, Stienstra R, van Riel PL, et al. (2010) Functional consequences of DECTIN-1 early stop codon polymorphism Y238X in rheumatoid arthritis. Arthritis Res Ther 12: R26.
[18]
Graham LM, Tsoni SV, Willment JA, Williams DL, Taylor PR, et al. (2006) Soluble Dectin-1 as a tool to detect beta-glucans. J Immunol Methods 314: 164–9.
[19]
Ujita M, Nagayama H, Kanie S, Koike S, Ikeyama Y, et al. (2009) Carbohydrate binding specificity of recombinant human macrophage beta-glucan receptor dectin-1. Biosci Biotechnol Biochem 73: 237–40.
Ostrosky-Zeichner L, Casadevall A, Galgiani JN, Odds FC, Rex JH (2010) An insight into the antifungal pipeline: selected new molecules and beyond. Nat Rev Drug Discov 9: 719–27.
[22]
Gantner BN, Simmons RM, Underhill DM (2005) Dectin-1 mediates macrophage recognition of Candida albicans yeast but not filaments. EMBO J 24: 1277–86.
[23]
Galán-Díez M, Arana DM, Serrano-Gómez D, Kremer L, Casasnovas JM, et al. (2010) Candida albicans beta-glucan exposure is controlled by the fungal CEK1-mediated mitogen-activated protein kinase pathway that modulates immune responses triggered through dectin-1. Infect Immun 78: 1426–36.
[24]
Kim NS, Byun TH, Lee GM (2001) Key determinants in the occurrence of clonal variation in humanized antibody expression of cho cells during dihydrofolate reductase mediated gene amplification. Biotechnol Prog 17: 69–75.
[25]
Wurm FM (2004) Production of recombinant protein therapeutics in cultivated mammalian cells. Nat Biotechnol 22: 1393–8.
[26]
Butler M (2005) Animal cell cultures: recent achievements and perspectives in the production of biopharmaceuticals. Appl Microbiol Biotechnol 68: 283–91.
[27]
Kaufman RJ, Sharp PA (1982) Amplification and expression of sequences cotransfected with a modular dihydrofolate reductase complementary dna gene. J Mol Biol 159: 601–21.
[28]
Urlaub G, K？s E, Carothers AM, Chasin LA (1983) Deletion of the diploid dihydrofolate reductase locus from cultured mammalian cells. Cell 33: 405–12.
[29]
Cacciatore JJ, Chasin LA, Leonard EF (2010) Gene amplification and vector engineering to achieve rapid and high-level therapeutic protein production using the Dhfr-based CHO cell selection system. Biotechnol Adv 28: 673–81.
[30]
Kim JY, Kim YG, Lee GM (2012) CHO cells in biotechnology for production of recombinant proteins: current state and further potential. Appl Microbiol Biotechnol 93: 917–30.
[31]
Ng SK, Wang DI, Yap MG (2007) Application of destabilizing sequences on selection marker for improved recombinant protein productivity in CHO-DG44. Metab Eng 9: 304–16.
[32]
Ghoda L, van Daalen Wetters T, Macrae M, Ascherman D, Coffino P (1989) Prevention of rapid intracellular degradation of ODC by a carboxyl-terminal truncation. Science 243: 1493–5.
[33]
Zubiaga AM, Belasco JG, Greenberg ME (1995) The nonamer is the key AU-rich sequence motif that mediates mRNA degradation. Mol Cell Biol 15: 2219–30.
[34]
Schilling J, Beverly S, Simonson C, Crouse G, Setzer D, et al. (1982) Dihydrofolate reductase gene structure and variable structure of amplified DNA structure in mouse cell lines. In: Schimke RT, editor. Gene Amplification. New York: Cold Spring Harbor Laboratory Press. pp. 149–153.
[35]
Milbrandt JD, Heintz NH, White WC, Rothman SM, Hamlin JL (1981) Methotrexate-resistant Chinese hamster ovary cells have amplified a 135-kilobase-pair region that includes the dihydrofolate reductase gene. Proc Natl Acad Sci U S A 78: 6043–7.
[36]
Ng SK, Lin W, Sachdeva R, Wang DI, Yap MG (2010) Vector fragmentation: characterizing vector integrity in transfected clones by Southern blotting. Biotechnol Prog 26: 11–20.
[37]
Kaufman RJ, Davies MV, Wasley LC, Michnick D (1991) Improved vectors for stable expression of foreign genes in mammalian cells by use of the untranslated leader sequence from EMC virus. Nucleic Acids Res 19: 4485–90.
[38]
Hobbs S, Jitrapakdee S, Wallace JC (1998) Development of a bicistronic vector driven by the human polypeptide chain elongation factor 1alpha promoter for creation of stable mammalian cell lines that express very high levels of recombinant proteins. Biochem Biophys Res Commun 252: 368–72.
[39]
Gurtu V, Yan G, Zhang G (1996) IRES bicistronic expression vectors for efficient creation of stable mammalian cell lines. Biochem Biophys Res Commun 229: 295–8.
[40]
Rees S, Coote J, Stables J, Goodson S, Harris S, et al. (1996) Bicistronic vector for the creation of stable mammalian cell lines that predisposes all antibiotic-resistant cells to express recombinant protein. Biotechniques 20: 102–10, 102-4, 106, 108-10.
[41]
Baird SD, Turcotte M, Korneluk RG, Holcik M (2006) Searching for IRES. RNA 12: 1755–85.
[42]
Jang SK, Kr？usslich HG, Nicklin MJ, Duke GM, Palmenberg AC, et al. (1988) A segment of the 5′ nontranslated region of encephalomyocarditis virus RNA directs internal entry of ribosomes during in vitro translation. J Virol 62: 2636–43.
[43]
Hoffman MA, Palmenberg AC (1995) Mutational analysis of the J-K stem-loop region of the encephalomyocarditis virus IRES. J Virol 69: 4399–406.
[44]
Bochkov YA, Palmenberg AC (2006) Translational efficiency of EMCV IRES in bicistronic vectors is dependent upon IRES sequence and gene location. Biotechniques 41: 283–4.
[45]
Aucouturier P, Khamlichi AA, Preud'homme JL, Bauwens M, Touchard G, et al. (1992) Complementary DNA sequence of human amyloidogenic immunoglobulin light-chain precursors. Biochem J 285: 149–52.
[46]
Ho SC, Bardor M, Feng H, Mariati, Tong YW, et al. (2012) IRES-mediated Tricistronic vectors for enhancing generation of high monoclonal antibody expressing CHO cell lines. J Biotechnol 157: 130–9.
[47]
Subiao L, Xiangming S, Yuanxing Z (2005) Insight into metabolism of CHO cells at low glucose concentration on the basis of the determination of intracellular metabolites. Process Biochemistry 40: 1917–1921.
[48]
Rajendra Y, Kiseljak D, Baldi L, Hacker DL, Wurm FM (2012) Reduced glutamine concentration improves protein production in growth-arrested CHO-DG44 and HEK-293E cells. Biotechnol Lett 34: 619–26.
[49]
Bollati-Fogolín M, Forno G, Nimtz M, Conradt HS, Etcheverrigaray M, et al. (2005) Temperature reduction in cultures of hGM-CSF-expressing CHO cells: effect on productivity and product quality. Biotechnol Prog 21: 17–21.
[50]
Schatz SM, Kerschbaumer RJ, Gerstenbauer G, Kral M, Dorner F, et al. (2003) Higher expression of Fab antibody fragments in a CHO cell line at reduced temperature. Biotechnol Bioeng 84: 433–8.
[51]
Rodrigues Goulart H, Arthuso Fdos S, Capone MV, de Oliveira TL, Bartolini P, et al. (2010) Enhancement of human prolactin synthesis by sodium butyrate addition to serum-free CHO cell culture. J Biomed Biotechnol 2010: 405872.
[52]
Jiang Z, Sharfstein ST (2008) Sodium butyrate stimulates monoclonal antibody over-expression in CHO cells by improving gene accessibility. Biotechnol Bioeng 100: 189–94.
[53]
Loignon M, Perret S, Kelly J, Boulais D, Cass B, et al. (2008) Stable high volumetric production of glycosylated human recombinant IFNalpha2b in HEK293 cells. BMC Biotechnol 8: 65.
[54]
Campbell M, Corisdeo S, McGee C, Kraichely D (2010) Utilization of site-specific recombination for generating therapeutic protein producing cell lines. Mol Biotechnol 45: 199–202.
[55]
Zhang Z, Yang X, Yang H, Yu X, Li Y, et al. (2011) New strategy for large-scale preparation of the extracellular domain of tumor-associated antigen HAb18G/CD147 (HAb18GED). J Biosci Bioeng 111: 1–6.
[56]
Westwood AD, Rowe DA, Clarke HR (2010) Improved recombinant protein yield using a codon deoptimized DHFR selectable marker in a CHEF1 expression plasmid. Biotechnol Prog 26: 1558–66.
[57]
Lucas BK, Giere LM, DeMarco RA, Shen A, Chisholm V, et al. (1996) High-level production of recombinant proteins in CHO cells using a dicistronic DHFR intron expression vector. Nucleic Acids Res 24: 1774–9.
[58]
Aldrich TL, Viaje A, Morris AE (2003) EASE vectors for rapid stable expression of recombinant antibodies. Biotechnol Prog 19: 1433–8.
[59]
Ye J, Alvin K, Latif H, Hsu A, Parikh V, et al. (2010) Rapid protein production using CHO stable transfection pools. Biotechnol Prog 26: 1431–7.
[60]
Kato Y, Adachi Y, Ohno N (2006) Contribution of N-linked oligosaccharides to the expression and functions of beta-glucan receptor, Dectin-1. Biol Pharm Bull 29: 1580–6.
[61]
Lee KG, Xu S, Kang ZH, Huo J, Huang M, et al. (2012) Bruton's tyrosine kinase phosphorylates Toll-like receptor 3 to initiate antiviral response. Proc Natl Acad Sci U S A 109: 5791–6.
