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.

Recent advances in mammalian protein production

“…The authors tested this strategy with the expression of interferon gamma (IFNγ) and found that 1.7‐, 6.6‐ and 13.3‐fold improvements in specific IFNγ productivities were obtained with the application of ARE, MODC PEST, and both ARE and MODC PEST, respectively. In an attempt to more closely link the gene of interest and selection marker (thereby preventing the possibility of gene fragmentation and loss of the target gene), Lam and colleagues introduced the dhfr gene directly downstream of the gene of interest using an Internal Ribosome Entry Sequence (IRES) [89]. This strategy allowed for both genes to be translated off of the same RNA; in addition, the stringency of selection was further increased by adding the PEST sequence to the dhfr gene.…”

Advances in Mammalian Cell Line Development Technologies for Recombinant Protein Production

“…This concept has been applied to improve the odds of picking a high producer cell clone even when gene amplification is used [29,34,35,36,37,38]. In a recent study, the application of IRES has also allowed for high recombinant protein production from MTX amplified cell pools, without the need for cloning [39]. …”

“…In addition, the use of AU-rich elements (ARE) and murine ornithine decarboxylase (MODC) PEST region as respective mRNA and protein destabilizing elements have been shown to successfully weaken the selection marker, which results in improvements in recombinant protein productivity using the MTX amplification system [42]. In a follow up study, an attenuated IRES element to reduce the expression of the downstream selection marker gene has also been employed to substitute ARE, resulting in high recombinant protein titers [39]. …”

Application of microRNA Targeted 3′UTRs to Repress DHFR Selection Marker Expression for Development of Recombinant Antibody Expressing CHO Cell Pools

“…For example, Wu et al used a short hairpin RNA targeted to the DHFR gene to generate recombinant IgG expressing CHO clones and found that this approach improved IgG expression by more than 100% and genomic stability in MTX free culture by 30% . The use of a destabilized‐DHFR selection marker linked to an attenuated IRES element has also been shown to be effective in generating high yielding recombinant CHO cell lines . The use of a codon de‐optimized DHFR selectable marker to improve selection stringency in the presence of MTX has also been shown to result in enhanced expression of recombinant proteins, showing that reducing translational efficiency of DHFR, and hence its expression, can be used to isolate cell lines with improved stringency and higher productivities without multiple and time‐consuming gene amplification steps …”