Hwang Su-Jeong, Kim Hak-Zoo, Koh Gou Young, Lee Gyun Min
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1 Kusong-Dong, Yuseong-Gu, Daejon 305-701, South Korea.
In Vitro Cell Dev Biol Anim. 2007 May-Jun;43(5-6):162-7. doi: 10.1007/s11626-007-9037-1. Epub 2007 Jul 10.
Angiopoietin-1 (Ang1) is an essential molecule for blood vessel formation. In an effort to produce large quantities of Ang1, recombinant Chinese hamster ovary (rCHO) cells expressing a high level of recombinant human Ang1 protein (rhAng1) with an amino terminal FLAG-tag were constructed by transfecting the expression vector into dihydrofolate reductase-deficient CHO cells and subsequent gene amplification in a medium containing step-wise increments of methotrexate, such as 0.02, 0.08, and 0.32 microM. The rhAng1 secreted from rCHO cells was purified at a purification yield of 18.4% from the cultured medium using an anti-FLAG M2 agarose affinity gel. SDS-PAGE and Western blot analyses showed that rCHO cells secret rhAng1 as heterogeneous multimers. Moreover, rhAng1 expressed in rCHO cells is biologically active in vitro as demonstrated by its ability to bind to the Tie2 receptor and to phosphorylate Tie2. Therefore, the rhAng1 produced from CHO cells could be useful for clarifying the biological effects of exogenous rhAng1 in the future.
血管生成素-1（Ang1）是血管形成的必需分子。为了大量生产Ang1，通过将表达载体转染到二氢叶酸还原酶缺陷型CHO细胞中，并随后在含有逐步增加甲氨蝶呤（如0.02、0.08和0.32微摩尔）的培养基中进行基因扩增，构建了表达高水平带有氨基末端FLAG标签的重组人血管生成素-1蛋白（rhAng1）的重组中国仓鼠卵巢（rCHO）细胞。使用抗FLAG M2琼脂糖亲和凝胶从培养基中纯化rCHO细胞分泌的rhAng1，纯化产率为18.4%。SDS-PAGE和蛋白质印迹分析表明，rCHO细胞分泌的rhAng1为异质性多聚体。此外，rCHO细胞中表达的rhAng1在体外具有生物学活性，这通过其与Tie2受体结合并使Tie2磷酸化的能力得以证明。因此，CHO细胞产生的rhAng1未来可能有助于阐明外源性rhAng1的生物学效应。
