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

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 mug/mL) of recombinant human Ang2 protein (rhAng2) with an amino-terminal FLAG-tag was constructed by transfecting the expression vectors into dihydrofolate reductase (dhft)-deficient CHO cells and the subsequent gene amplification in medium containing step-wise increments in methotrexate level such as 0.02, 0.08, and 0.32 muM. 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. (C) 2004 Elsevier Inc. All rights reserved.