Expression, Purification and Characterization of Recombinant Human Angiogenin in Pichia pastoris
Expression, Purification and Characterization of Recombinant Human Angiogenin in Pichia pastoris
复制标题
DOI：
10.1271/bbb.120178
复制
发表时间：
2012-07
期刊：
影响因子：
--
通讯作者：
W. Xia;Wen-liang Fu;L. Cai;Xin Cai;Yuan-yuan Wang;M. Zou;Dong-Gang Xu
中科院分区：
文献类型：
--
作者：
W. Xia;Wen-liang Fu;L. Cai;Xin Cai;Yuan-yuan Wang;M. Zou;Dong-Gang Xu
血管生成素（angiogenin，Ang）在诱导血管生成、促进细胞增殖和抑制细胞凋亡方面具有重要作用，因此其临床应用潜力日益受到重视。为了获得可溶性、正确折叠的重组蛋白，将编码人血管紧张素（Ang）的DNA片段插入真核表达载体pPIC 9，转化毕赤酵母（Pichia pastoris）。重组人血管紧张素（rhAng）的表达量约占总分泌蛋白的70%。通过用SP Sepharose FF柱的色谱法从培养上清液中纯化Ang，得到30 mg/L，纯度为90%。生物学实验表明，rhAng可诱导新血管形成，促进HeLa细胞增殖，增加Erk 1/2磷酸化，上调c-myc表达。rhAng的制备为进一步的功能研究奠定了基础，并为大规模生产可溶性人Ang提供了有效的策略。
The potential of angiogenin (Ang) for clinical use has been highlighted in view of its important roles in inducing angiogenesis, facilitating cell proliferation, and inhibiting cell apoptosis. To produce soluble, correctly folded recombinant protein with a high yield, a DNA fragment encoding human Ang was inserted into eukaryotic expression vector pPIC9 and transformed into Pichia pastoris. The expression of recombinant human Ang (rhAng) accounted for about 70% of total secreted proteins. Purifying the Ang from the culture supernatant yielded 30 mg/L at 90% purity by chromatography with a SP Sepharose FF column. Biological assays indicated that rhAng can induce new blood-vessel formation, promote HeLa cell proliferation, increase Erk1/2 phosphorylation, and upregulate c-myc expression. Preparation of bioactive rhAng might lay the basis for further functional study, and might provide an effective strategy for large-scale production of soluble human Ang.