title: High-throughput sorting of the highest producing cell via a transiently protein-anchored system.
abstract: Developing a high-throughput method for the effecient selection of the highest producing cell is very important for the production of recombinant protein drugs. Here, we developed a novel transiently protein-anchored system coupled with fluorescence activated cell sorting (FACS) for the efficient selection of the highest producing cell. A furin cleavage peptide (RAKR) was used to join a human anti-epithelial growth factor antibody (αEGFR Ab) and the extracellular-transmembrane-cytosolic domains of the mouse B7-1 antigen (B7). The furin inhibitor can transiently switch secreted αEGFR Ab into a membrane-anchored form. After cell sorting, the level of membrane αEGFR Ab-RAKR-B7 is proportional to the amount of secreted αEGFR Ab in the medium. We further selected 23 αEGFR Ab expressing cells and demonstrated a high correlation (R2 = 0.9165) between the secretion level and surface expression levels of αEGFR Ab. These results suggested that the novel transiently protein-anchored system can easily and efficiently select the highest producing cells, reducing the cost for the production of biopharmaceuticals.
authors.affiliation: Graduate Institute of Pharmacognosy, Taipei Medical University, Taipei, Taiwan; Ph.D. Program for Clinical Drug Discovery from Botanical Herbs, Taipei Medical University, Taipei, Taiwan; Master Program for Clinical Pharmacogenomics and Pharmacoproteomics, Taipei Medical University, Taipei, Taiwan.
authors.forename: Kuo-Hsiang
authors.initials: KH
authors.lastname: Chuang
authors.affiliation: Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.
authors.forename: Yuan-Chin
authors.initials: YC
authors.lastname: Hsieh
authors.affiliation: Department of Biomedical Science and Environmental Biology, Kaohsiung Medical University, Kaohsiung, Taiwan.
authors.forename: I-Shiuan
authors.initials: IS
authors.lastname: Chiang
authors.affiliation: Department of Biomedical Science and Environmental Biology, Kaohsiung Medical University, Kaohsiung, Taiwan.
authors.forename: Chih-Hung
authors.initials: CH
authors.lastname: Chuang
authors.affiliation: Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.
authors.forename: Chien-Han
authors.initials: CH
authors.lastname: Kao
authors.affiliation: Graduate Institute of Pharmacognosy, Taipei Medical University, Taipei, Taiwan.
authors.forename: Ta-Chun
authors.initials: TC
authors.lastname: Cheng
authors.affiliation: Department of Biochemistry, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.
authors.forename: Yeng-Tseng
authors.initials: YT
authors.lastname: Wang
authors.affiliation: Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan.
authors.forename: Wen-Wei
authors.initials: WW
authors.lastname: Lin
authors.affiliation: Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan.
authors.forename: Bing-Mae
authors.initials: BM
authors.lastname: Chen
authors.affiliation: Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan.
authors.forename: Steve R
authors.initials: SR
authors.lastname: Roffler
authors.affiliation: Department of Radiation Oncology, Cancer Center, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan.
authors.forename: Ming-Yii
authors.initials: MY
authors.lastname: Huang
authors.affiliation: Department of Biomedical Science and Environmental Biology, Kaohsiung Medical University, Kaohsiung, Taiwan; Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan; Cancer Center, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan.
authors.forename: Tian-Lu
authors.initials: TL
authors.lastname: Cheng
mesh_terms: D000818:Animals; D000906:Antibodies / Q000378:metabolism; D018122:B7-1 Antigen / Q000378:metabolism; D002460:Cell Line; D002469:Cell Separation / Q000379:methods*; D003600:Cytosol / Q000378:metabolism; D066246:ErbB Receptors / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods*; D045683:Furin / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D011485:Protein Binding / Q000502:physiology; D021381:Protein Transport / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000378:metabolism
chemical_list: D000906:Antibodies; D018122:B7-1 Antigen; D011993:Recombinant Fusion Proteins; D066246:ErbB Receptors; D045683:Furin