Krebswissenschaft und -therapie

Validation of Different Systems for Tumstatin Expression and its in-vitro and in-vivo Activities

Abstract

Chandra S Boosani, Ashok K Varma and Akulapalli Sudhakar

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 ?3 chain Type IV collagen, a3(IV)NC1 (tumstatin) was isolated from human placental tissue and cloned in three different expression vectors pET22b, pcBFT and pAcHLTA 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.