Expression and purification of recombinant human angiopoietin-4 produced in Chinese hamster ovary cells

Abstract

The angiopoietin (Ang) family of growth factors plays a role in both blood vessel sprouting and blood vessel regression. And it includes Angl, Ang2, Ang3, and Ang4, all of which bind to the endothelial receptor tyrosine kinase Tie2. Ang4 (human) is interspecies ortholog and an agonist of Tie2. Recombinant Chinese hamster ovary (rCHO) cell lines expressing a high level (7 ㎍/mL) of recombinant human Ang4 protein (rhAng4) 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 rhAng4 secreted from rCHO cells was purified at a purification yield of 53.3% from the cultured medium using an anti-FLAG M2 agarose affinity gel. SDS-PAGE and Western blot analyses showed that rCHO cells secret rhAng4 as a dominantly homodimeric glycoprotein form. Furthermore, rhAng4 binds to the Tie2 receptor and phosphortlates Tie2 in a concentration-dependent manner. Therefore, our rhAng4 could be useful for clarifying biological effect of exogenous Ang4 in future.