Validation of Different Systems for Tumstatin Expression and its in-vitro and iv-vivo Activities.
Validation of Different Systems for Tumstatin Expression and its in-vitro and iv-vivo Activities.
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DOI：
10.4172/1948-5956.1000002
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发表时间：
2009-11
期刊：
影响因子：
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通讯作者：
Chandra S Boosani;A. Varma;A. Sudhakar
中科院分区：
文献类型：
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作者：
Chandra S Boosani;A. Varma;A. Sudhakar
本研究的目的是确定一个有效的表达系统，用于重组抗血管生成蛋白tumstatin的纯化。从人胎盘组织中分离编码α 3链IV型胶原的羧基末端非胶原结构域的序列，α 3（IV）NC 1（肿瘤抑制素），并克隆到三种不同的表达载体pET 22 b、pcBFT和pAcHLT-A中，分别在细菌、哺乳动物和Sf-9昆虫细胞中表达。通过考马斯亮蓝染色和免疫印迹法对tumstatin的表达和纯化情况进行评估，并基于可溶性蛋白的产量来确定效率。我们的研究结果表明，杆状病毒表达系统是有效的可扩展产量的可溶性蛋白，可以纯化其生物活性形式。利用杆状病毒表达的tumstatin进行了抗血管生成和抗肿瘤功能的研究，如抑制内皮细胞增殖、细胞活力、迁移、管形成、帽依赖性蛋白翻译以及相关的信号传导机制，包括体内肿瘤研究。与两种表达系统相比，我们评估的使用修饰的杆状病毒表达系统的方法显示出高表达和高产量的生物活性tumstatin，表明杆状病毒表达系统是用于临床前和临床试验所需的可溶性tumstatin的批量生产的理想方法。
The aim of the present study is to identify an effective and efficient expression system for purification of recombinant antiangiogenic protein tumstatin. The sequence encoding carboxy-terminal non-collagenous domain of α3 chain Type IV collagen, α3(IV)NC1 (tumstatin) was isolated from human placental tissue and cloned in three different expression vectors pET22b, pcBFT and pAcHLT-A to express it in bacteria, mammalian and Sf-9 insect cells respectively. Expression and purification profiles of tumstatin were evaluated by coomassie staining and immunoblotting, and the efficiency was determined based on the yields of soluble protein. Our results indicate that, baculovirus expression system was efficient for scalable yields of soluble protein that could be purified in its biologically active form. This baculovirus expressed tumstatin was used to evaluate its anti-angiogenic and anti-tumarogenic functions such as inhibition of endothelial cell proliferation, cell viability, migration, tube formation, cap dependent protein translation and the associated signaling mechanism including in-vivo tumor study. Our evaluated approaches using a modified baculovirus expression system shows high expression and high yield of biologically active tumstatin, as compared to two expression systems, indicating baculovirus expression system to be an ideal method for bulk production of soluble tumstatin that needed for pre-clinical and clinical trails.