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High-level Expression and Purification of a Designed Angiopoietin-1 Chimeric Protein, COMP-Ang1, Produced in Chinese Hamster Ovary Cells

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Abstract

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 μg/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 μM. 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.

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Notes

  1. Ang1, Angiopoietin-1.

  2. COMP-Ang1, the short coiled-coil domain of cartilage oligomeric matrix protein of modified Ang1.

  3. rCHO, recombinant Chinese hamster ovary.

  4. CMV, cytomegalovirus.

  5. dhfr, dihydrofolate reductase.

  6. dFBS, dialyzed fetal bovine serum.

  7. MTX, methotrexate.

  8. HUVECs, human umbilical vein endothelial cells.

Abbreviations

Ang1:

Angiopoietin-1

COMP-Ang1:

The short coiled-coil domain of cartilage oligomeric matrix protein of modified Ang1

rCHO:

Recombinant Chinese hamster ovary

CMV:

Cytomegalovirus

dhfr :

Dihydrofolate reductase

dFBS:

Dialyzed fetal bovine serum

MTX:

Methotrexate

HUVECs:

Human umbilical vein endothelial cells

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Acknowledgments

This work was supported in part by grants from the Ministry of Commerce, Industry, and Energy and Daejon City (Bio/RIS program), the Ministry of Science and Technology (National Research Laboratory Programs, 2004-02376, GYK), and the Ministry of Education (Brain Korea 21 Program).

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Authors and Affiliations

  1. Department of Biological Sciences, KAIST, 373-1 Kusong-Dong, Yuseong-gu, Daejon, 305-701, Korea

    Su-Jeong Hwang, Sung Hyun Kim, Hak-Zoo Kim, Gou Young Koh & Gyun Min Lee

  2. Structural Biology, Paul Scherrer Institute, CH-5232, Villigen PSI, Switzerland

    Michel O. Steinmetz

Authors
  1. Su-Jeong Hwang
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  2. Sung Hyun Kim
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  3. Hak-Zoo Kim
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  4. Michel O. Steinmetz
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  5. Gou Young Koh
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  6. Gyun Min Lee
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Corresponding author

Correspondence to Gyun Min Lee.

Additional information

S.-J. Hwang and S. H. Kim are contributed equally to this work.

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Hwang, SJ., Kim, S.H., Kim, HZ. et al. High-level Expression and Purification of a Designed Angiopoietin-1 Chimeric Protein, COMP-Ang1, Produced in Chinese Hamster Ovary Cells. Protein J 27, 319–326 (2008). https://doi.org/10.1007/s10930-008-9140-5

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  • DOI: https://doi.org/10.1007/s10930-008-9140-5

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