title: Production of functional soluble Dectin-1 glycoprotein using an IRES-linked destabilized-dihydrofolate reductase expression vector.
abstract: 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.
authors.affiliation: Bioprocessing Technology Institute, Agency for Science, Technology and Research-A*STAR, Singapore, Singapore. ng_say_kong@bti.a-star.edu.sg
authors.forename: Say Kong
authors.initials: SK
authors.lastname: Ng
authors.forename: Tessa Rui Min
authors.initials: TR
authors.lastname: Tan
authors.forename: Yang
authors.initials: Y
authors.lastname: Wang
authors.forename: Daniel
authors.initials: D
authors.lastname: Ng
authors.forename: Lin-Tang
authors.initials: LT
authors.lastname: Goh
authors.forename: Muriel
authors.initials: M
authors.lastname: Bardor
authors.forename: Victor Vai Tak
authors.initials: VV
authors.lastname: Wong
authors.forename: Kong Peng
authors.initials: KP
authors.lastname: Lam
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000935:Antifungal Agents / Q000737:chemistry / Q000378:metabolism; D019149:Bioreactors; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D005822:Genetic Vectors; D006023:Glycoproteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D037181:Lectins, C-Type / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D008727:Methotrexate / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D010442:Peptide Chain Initiation, Translational; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D012995:Solubility; D013762:Tetrahydrofolate Dehydrogenase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D015533:Transcriptional Activation / Q000187:drug effects; D015054:Zymosan / Q000737:chemistry; D000099181:Dectin-1
chemical_list: D000935:Antifungal Agents; D006023:Glycoproteins; D037181:Lectins, C-Type; D008727:Methotrexate; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D013762:Tetrahydrofolate Dehydrogenase; D015054:Zymosan; D000099181:Dectin-1