Selecting cells expressing high levels of recombinant proteins using the GPI-anchored protein with selenocysteine system
Section snippets
Cell culture and reagents
HEK293 cells were cultured in Dulbecco's Modified Eagle's Medium supplemented with 10% fetal calf serum. CHO–K1 cells were cultured in Dulbecco's Modified Eagle's Medium/Ham's Nutrient Mixture F-12 supplemented with 10% fetal calf serum. Puromycin (1 μg/ml) and streptomycin/penicillin (1 μg/ml) were used where necessary. Mouse monoclonal anti-CD59 (clone 5H8), which was kindly provided by Taroh Kinoshita (Osaka University); rabbit monoclonal anti-GAPDH (Abcam, Cambridge, UK); mouse monoclonal
Recombinant LIPA can be expressed on the cell surface using the GPS system
Our aim was to develop a tool for isolating cells expressing high levels of recombinant proteins from a large cell population. First, we designed a new construct that has the Sec insertion sequence (SECIS) in the 3′-UTR and an in-frame UGA codon between the open reading frame (ORF) and GPI-attachment signal, generating ORF-UGA-GPI-UAA-SECIS. It has been reported that Sec is incorporated into approximately 4–10% of protein molecules, whereas the UGA codon is recognized as a stop codon in the
Discussion
Over 2000 human proteins are secreted proteins, including hormones, antibodies, cytokines, and growth factors (29). Hundreds of these proteins are being developed as recombinant proteins for preclinical and clinical purposes. Currently, to increase productivity, gene amplification systems based on either methotrexate amplification technology or glutamine synthetase system are frequently used (30,31). However, despite important advances in these methods, the rapid selection of cells expressing
Acknowledgments
This work was supported by grants-in-aid from the National Natural Science Foundation of China (31900923 and 31770853), the Program of Introducing Talents of Discipline to Universities (111-2-06), National first-class discipline program of Light Industry Technology and Engineering (LITE2018-015), Top-notch Academic Programs Project of Jiangsu Higher Education Institutions, and the International Joint Research Laboratory for Investigation of Glycoprotein Biosynthesis at Jiangnan University.
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Present Address: Institute of Microbiology, ETH Zürich, Vladimir-Prelog-Weg 1-5/10, 8093 Zürich, Switzerland.