Abstract
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.
Highlights
Vascular basement membrane (VBM) is an important constituent of blood vessels providing structural support (Darland and D’Amore, 1999)
At present there are about 25 endogenous angioinhibitors in clinical trials and many more in preclinical studies for the treatment of cancer. These angioinhibitors fall into two general categories: (a) antibodies or small molecules that target pro-angiogenic factors of tumor cells such as VEGF, bFGF or PDGF, and (b) endogenous angioinhibitors such as thrombopondin-1, angiostatin, interferons, endostatin and some of the non collagenous (NC1) domains of Type IV collagen that target vascular endothelial cells (Boosani et al, 2007; O’Reilly et al, 1997; O’Reilly et al, 1994; Petitclerc et al, 2000; Qian et al, 1997; Sudhakar and Boosani, 2007; Sudhakar and Boosani, 2008; Sudhakar et al, 2005)
We previously reported that tumstatin binds to αVβ3/α3β1 integrins and inhibits PI3K/Akt/mTOR/ 4E-BP1/COX-2 signaling leading to the inhibition of protein synthesis and hypoxic tumor angiogenesis (Boosani et al, 2007; Maeshima et al, 2002; Sudhakar et al, 2003)
Summary
Vascular basement membrane (VBM) is an important constituent of blood vessels providing structural support (Darland and D’Amore, 1999). At present there are about 25 endogenous angioinhibitors in clinical trials and many more in preclinical studies for the treatment of cancer These angioinhibitors fall into two general categories: (a) antibodies or small molecules that target pro-angiogenic factors of tumor cells such as VEGF, bFGF or PDGF, and (b) endogenous angioinhibitors such as thrombopondin-1, angiostatin, interferons, endostatin and some of the non collagenous (NC1) domains of Type IV collagen that target vascular endothelial cells (Boosani et al, 2007; O’Reilly et al, 1997; O’Reilly et al, 1994; Petitclerc et al, 2000; Qian et al, 1997; Sudhakar and Boosani, 2007; Sudhakar and Boosani, 2008; Sudhakar et al, 2005). This novel discovery of NC1 domains from human Type IV collagen as angioinhibitors initiated a new line of research in several laboratories and identified their significance for the treatment of cancer (Boosani et al, 2007; Boosani and Sudhakar, 2006; Borza et al, 2006; Maeshima et al, 2002; Marneros and Olsen, 2001; Petitclerc et al, 2000; Roth et al, 2005; Sudhakar and Boosani, 2007; Sudhakar et al, 2005; Sudhakar et al, 2003)
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