Expression and purification of recombinant human angiopoietin-2 produced in Chinese hamster ovary cells.
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 microg/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 microM. 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.
Hwang
Su-Jeong
SJ
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1 Kusong-Dong, Yusong-gu, Daejon 305-701, Republic of Korea.
Choi
Han Ho
HH
Kim
Kyung-Tae
KT
Hong
Hyo Jeong
HJ
Koh
Gou Young
GY
Lee
Gyun Min
GM
D042702:Angiopoietin-2 / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D002470:Cell Survival; D006224:Cricetinae; D003412:Cricetulus; D017077:Culture Media, Conditioned / Q000032:analysis; D016895:Culture Media, Serum-Free / Q000032:analysis; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005784:Gene Amplification; D005822:Genetic Vectors; D006801:Humans; D008727:Methotrexate / Q000494:pharmacology; D010957:Plasmids; D042810:Receptor, TIE-1 / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000172:deficiency; D014162:Transfection
D042702:Angiopoietin-2; D017077:Culture Media, Conditioned; D016895:Culture Media, Serum-Free; D011994:Recombinant Proteins; D013762:Tetrahydrofolate Dehydrogenase; D042810:Receptor, TIE-1; D008727:Methotrexate