High-level expression and purification of a designed angiopoietin-1 chimeric protein, COMP-Ang1, produced in Chinese hamster ovary cells.
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 microg/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 microM. 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.
Hwang
Su-Jeong
SJ
Department of Biological Sciences, KAIST, 373-1 Kusong-Dong, Yuseong-gu, Daejon 305-701, Korea.
Kim
Sung Hyun
SH
Kim
Hak-Zoo
HZ
Steinmetz
Michel O
MO
Koh
Gou Young
GY
Lee
Gyun Min
GM
D020816:Amino Acid Motifs; D042683:Angiopoietin-1 / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D015870:Gene Expression / Q000235:genetics*; D010766:Phosphorylation; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D042683:Angiopoietin-1; D011994:Recombinant Proteins