Laboratory of Molecular Genetics, Beijing Institute of Basic Medical Sciences, 100850, Beijing, China.
Mol Biol Rep. 2010 Dec;37(8):3909-13. doi: 10.1007/s11033-010-0047-9. Epub 2010 Mar 13.
Angiopoietin-2 (Ang2) is a member of the Ang family. Its potential in clinical use has been highlighted for its important roles in angiogenesis during the individual development and the growth of tumors. Ang2 is difficult to be expressed in E. coli for its unique structure. The expressions of Ang2 in insect cells (Sf9) and Chinese hamster ovary (CHO) cell line have been reported, however, the large-scale production of Ang2 for application is still pendent. In this study, the expression of Ang2 in Pichia pastoris expression system was described for the first time. The cDNA encoding Ang2 was cloned from the rat vascular tissue by RT-PCR, and inserted in the eukaryotic expression vector pPIZαA, and then transformed into P. pastoris KM71H cells. The expression of recombinant rat Ang2 (rrAng2) was induced by methanol and accounted for about 75% of the total secreted proteins. The recombinant protein was subsequently purified by HisTrap FF crude with a purity of 90%. Functional analysis of the purified rrAng2 demonstrated a specific activity in promoting the survival of ECV304 cells and binding to the Tie2 receptor. Preparation of bioactive rrAng2 not only lays the basis for further functional study but also provides a new strategy for soluble and large-scale production of human Ang2.
血管生成素 2（Ang2）是血管生成素家族的一员。由于其在个体发育和肿瘤生长过程中血管生成中的重要作用，其在临床应用中的潜力已得到强调。由于其独特的结构，Ang2 难以在大肠杆菌中表达。已经报道了 Ang2 在昆虫细胞（Sf9）和中国仓鼠卵巢（CHO）细胞系中的表达，然而，大规模生产 Ang2 用于应用仍有待研究。本研究首次描述了 Ang2 在毕赤酵母表达系统中的表达。通过 RT-PCR 从大鼠血管组织中克隆编码 Ang2 的 cDNA，并将其插入真核表达载体 pPIZαA，然后转化到毕赤酵母 KM71H 细胞中。重组大鼠 Ang2（rrAng2）的表达通过甲醇诱导，占总分泌蛋白的约 75%。重组蛋白随后通过 HisTrap FF 粗纯化，纯度为 90%。纯化的 rrAng2 的功能分析表明，其具有促进 ECV304 细胞存活和与 Tie2 受体结合的特异性活性。生物活性 rrAng2 的制备不仅为进一步的功能研究奠定了基础，也为可溶性和大规模生产人 Ang2 提供了新策略。
