Expression and purification of recombinant human angiopoietin-2 produced in Chinese hamster ovary cells

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Title
Expression and purification of recombinant human angiopoietin-2 produced in Chinese hamster ovary cells
Author(s)
S J Hwang; H H Choi; K T Kim; Hyo Jeong Hong; G Y Koh; G M Lee
Bibliographic Citation
Protein Expression and Purification, vol. 39, no. 2, pp. 175-183
Publication Year
2005
Abstract
Angiopoietin-2 (Ang2) is a complex regulator of vascular remodeling that plays a role in both blood vessel sprouting and blood vessel regression through its receptor Tie2. Recombinant Chinese hamster ovary (rCHO) cell lines expressing a high level (20 μg/mL) of recombinant human Ang2 protein (rhAng2) with an amino-terminal FLAG-tag was constructed by transfecting the expression vectors into dihydrofolate reductase (dhfr)-deficient CHO cells and the subsequent gene amplification in medium containing stepwise increments in methotrexate level such as 0.02, 0.08, and 0.32 μM. The rhAng2 secreted from rCHO cells was purified at a purification yield of 53.6% from the cultured medium using an anti-FLAG M2 agarose affinity gel. SDS-PAGE and Western blot analyses showed that rCHO cells secret rhAng2 as a homodimeric glycoprotein form. Furthermore, rhAng2 binds to the Tie2 receptor and phosphorylates Tie2 in a concentration-dependent manner. Therefore, our rhAng2 could be useful for clarifying biological effect of exogenous Ang2 in the future.
Keyword
angiopoietinchinese hamster ovary cellsFLAG-tag
ISSN
1046-5928
Publisher
Elsevier
DOI
http://dx.doi.org/10.1016/j.pep.2004.09.005
Type
Article
Appears in Collections:
1. Journal Articles > Journal Articles
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