High-level Expression and Purification of a Designed Angiopoietin-1 Chimeric Protein,COMP-Ang1, Produced in Chinese Hamster Ovary Cells |
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Authors: | Su-Jeong Hwang Sung Hyun Kim Hak-Zoo Kim Michel O. Steinmetz Gou Young Koh Gyun Min Lee |
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Affiliation: | Department of Biological Sciences, KAIST, 373-1 Kusong-Dong, Yuseong-gu, Daejon, 305-701, Korea. |
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Abstract: | A designed angiopoietin-1 (Ang1) chimeric protein with nonleaky angiogenic activity, COMP-Ang1, is an effective alternative to native Ang1 for therapeutic angiogenesis in vivo. Recombinant Chinese hamster ovary (rCHO) cell lines expressing a high level (>20 mug/mL) of COMP-Ang1 and an amino-terminal FLAG-tag were constructed by transfecting the expression vector into dihydrofolate reductase-deficient CHO cells and the subsequent gene amplification in medium containing stepwise increments in methotrexate level such as 0.02, 0.08, 0.32, and 1 muM. The COMP-Ang1 secreted from rCHO cells was purified at a purification yield of 40.3% from the culture medium using an anti-FLAG M2 agarose affinity gel. SDS-PAGE and Western blot analyses showed that rCHO cells secrete COMP-Ang1 in homopentameric and homotetrameric glycoprotein forms. Furthermore, COMP-Ang1 binds to the Tie2 receptor and phosphorylates Tie2, indicating its potential for therapeutic angiogenesis. |
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Keywords: | Angiopoietin COMP-Ang1 FLAG-tag Tie2 receptor Chinese hamster ovary cells |
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