Cloning, expression and purification of recombinant BMP-2 as a tool to evaluate the role of receptor B1 kinin with osteoblast differentiation induced by BMP-2

• Grant number: 12/07666-1
• Support Opportunities: Scholarships in Brazil - Master
• Start date: October 01, 2012
• End date: March 31, 2014
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: João Bosco Pesquero
• Grantee: Marina Rodrigues e Silva
• Host Institution: Escola Paulista de Medicina (EPM). Universidade Federal de São Paulo (UNIFESP). Campus São Paulo. São Paulo , SP, Brazil

Abstract

Bone tissue is a dynamic, remodeled continuously, tissue that depends on several factors for maintaining the balance between bone formation and resorption. Damages of this tissue are a serious public health problem. Millions of Brazilians suffer today with problems related to musculoskeletal injuries. The bone tissue is affected by several injuries among which highlight congenital metabolic diseases, as well as external factors such as fractures. All these commitments are interesting in finding a mechanism to repair damaged bone tissue. Currently the Tissue Engineering Techniques have been indicated in the treatment of lesions in bone tissue, being one of the most promising alternative therapies to repair bone damage. There are several mediators involved in osteogenic differentiation, which seek to repair and form bone tissue, one of these mediators are the bone morphogenetic proteins (BMPs). In addition to the mediators such as BMPs, some systems are related to the process of bone differentiation such as kallikrein-kinin system, this system has been associated with the destruction of bone matrix, but little known at the time of the real part of various system components in a pathological process. With the expression of recombinant BMP-2 and its use in the induction of bone differentiation process, this paper seeks to assist in understanding the B1 receptor involvement in the process of differentiation. To achieve this goal, the protein BMP-2 will be cloned from a cloning strategy that allows transfer of heterologous DNA sequences between vectors. This system was developed for the expression of recombinant protein fused to a histidine tail portion in its N or C-terminal, capable of binding affinity columns.The fusing of the histidines on the tail allows for easy purification of the protein using affinity chromatography. After cloning we evaluate the functional viability of the protein for use in tests of differentiation aimed at assessing the role of kinin B1 receptor in osteogenesis in vitro and prospect for further evaluation in vivo.