High-throughput sorting of the highest producing cell via a transiently protein-anchored system
- Resource Type
- Authors
- Chih-Hung Chuang; Steve R. Roffler; Tian-Lu Cheng; Ming-Yii Huang; Wen-Wei Lin; I-Shiuan Chiang; Chien-Han Kao; Kuo-Hsiang Chuang; Ta-Chun Cheng; Yuan-Chin Hsieh; Yeng-Tseng Wang; Bing-Mae Chen
- Source
- PLoS ONE, Vol 9, Iss 7, p e102569 (2014)
PLoS ONE
- Subject
- Protein Expression
Cell
Cell Separation
Protein Engineering
Biochemistry
Mice
Cytosol
Macromolecular Engineering
Furin
Multidisciplinary
biology
medicine.diagnostic_test
Cell sorting
Flow Cytometry
Recombinant Proteins
Cell biology
ErbB Receptors
Protein Transport
medicine.anatomical_structure
B7-1 Antigen
Engineering and Technology
Medicine
Protein Binding
Research Article
Biotechnology
Recombinant Fusion Proteins
Science
Bioengineering
Antibodies
Cell Line
Flow cytometry
Gene Expression and Vector Techniques
medicine
Animals
Humans
Secretion
Molecular Biology Techniques
Molecular Biology
Molecular Biology Assays and Analysis Techniques
HEK 293 cells
Biology and Life Sciences
Proteins
HEK293 Cells
Secretory protein
Synthetic Bioengineering
Cell culture
biology.protein
- Language
- English
- ISSN
- 1932-6203
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.