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Expression, purification and characterization of bone morphogenetic protein (BMP-2) for application in composites biomaterials with poly(E-caprolactone)

Grant number: 16/24724-6
Support type:Research Grants - Innovative Research in Small Business - PIPE
Duration: September 01, 2017 - August 31, 2019
Field of knowledge:Biological Sciences - Biochemistry
Principal Investigator:Paolo Bartolini
Grantee:Paolo Bartolini
Company:Laboratório Biosintesis P & D do Brasil Ltda
City: São Paulo
Co-Principal Investigators:Fabiana Medeiros da Silva
Assoc. researchers:João Ezequiel de Oliveira ; Miriam Fussae Suzuki ; Renata Damiani
Associated research grant:15/15446-0 - Expression, purification and characterization of bone morphogenetic protein (BMP-2) for application in composites biomaterials with poly(E-caprolactone), AP.PIPE
Associated scholarship(s):17/21982-7 - Expression, purification and characterization of bone morphogenetic protein (BMP-2) for application in composites biomaterials with poly(E-caprolactone), BP.PIPE

Abstract

The goal of the present Project, whose Phase-1 will be completed in January 31st 2017 and whose partial Technical Report (first 6 months) has been already submitted to FAPESP, is the national synthesis and characterization of recombinant human bone morphogenetic protein-2 (rhBMP-2). During Phase-1, in agreement with the submitted proposal of technical viability, for the first time the periplasmic expression of hBMP-2 was demonstrated in Escherichia coli, through preliminary tests of identity, purity and potency. The latter, determined through an in vitro bioassay in myoblastic cells C2C12, has proven that the synthesized protein is bioactive, with a probable potency >90%, against a standard of E.coli cytoplasmic hBMP-2, distributed by GenScript (Piscataway, NJ, USA). The approximate yields were of the order of 1 µg/ml/A600, which allowed a first purification and characterization of the protein.The Phase-2 of the Project is planning to obtain a significant increase of the production yields, with a biomass of the order of 30-60 A600 units and expression yields of at least 2-4 µg/ml/A600. This will be carried out by utilizing different expression vectors, bacterial strain and, especially, bioreaction conditions under highly controlled parameters, in a 20-Liter Laboratory Bioreactor (MBR, Zurich, Suiça) and, possibly, also in an industrial/pilot bioreactor of 150 Liters. Another goal will be the setting up of a purification (downstream) process with yields of the order of 40-60 %, and purity > 95%. Our company (Biosíntesis) already holds the technological platform and the intellectual property to obtain the polymer poly(µ-caprolactone) (PCL) and consequent preparation of biologically active composites. In this Phase-2 we will therefore obtain and tested different composites, formed by PCL and rhBMP-2 and by PCL and human growth hormone (hGH). The incorporation of the active compounds rhBMP-2 and hGH will provide prototypes and formulations for comparative osteoinductive studies. Human growth hormone is a protein on which the research group that is developing this project has an extensive technological know-how, with several publications and a patent deposited by the Biotechnology Center of the IPEN. This hormone, moreover, already demonstrated a clear osteogenic activity. The osteoinductive effects typical of rhBMP-2, will be therefore tested in comparison with hGH in an animal model to study Osteointegration efficiency and also in a model of Osteogenesis Imperfecta (OI). The latter model is being developed in the animal house of our biotechnology center (CB), the mutant breeding stocks ("oim" mice) having been acquired from the Jackson Laboratory (USA), thanks to a grant from FAPESP, related to Growth Hormone Gene Therapy activities of the same CB-IPEN (Proc. 2014/19757-7). (AU)