Validation of Different Systems for Tumstatin Expression and its in-vitro and in-vivo Activities
- *Corresponding Author:
- Dr. Sudhakar Akulapalli
Assistant Professor/ Staff Scientist II
Creighton University School of Medicine and University of Nebraska Medical Center,
Director & Coordinator: Cell Signaling and Tumor Angiogenesis Laboratory,
Boys Town National Research Hospital, 555 N, 30th Street
Omaha NE 68131,
Tel: (402) 498-6681, (402) 498-6322
Fax: (402) 498-6331
E-mail: [email protected]
Received Date: November 19, 2009; Accepted November 27, 2009; Online published November 27, 2009; Published July 02, 2010
Citation: Boosani CS, Varma AK, Sudhakar A (2009) Validation of Different Systems for Tumstatin Expression and its in-vitro and in-vivo Activities. J Cancer Sci Ther 1: 008-018. doi:10.4172/1948-5956.1000002
Copyright: © 2009 Boosani CS, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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.