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Characterization of intermediate states of protein folding and obtainment of biofunctional proteins by high hydrostatic pressure

Grant number: 10/13353-0
Support type:Regular Research Grants
Duration: December 01, 2010 - November 30, 2012
Field of knowledge:Biological Sciences - Biochemistry - Chemistry of Macromolecules
Principal Investigator:Ligia Ely Morganti Ferreira Dias
Grantee:Ligia Ely Morganti Ferreira Dias
Home Institution: Instituto de Pesquisas Energéticas e Nucleares (IPEN). Secretaria de Desenvolvimento Econômico (São Paulo - Estado). São Paulo , SP, Brazil

Abstract

Many relevant therapeutic proteins are found naturally in low concentrations. Escherichia coli is a very useful microorganism for recombinant protein production for structural and functional studies and for production of therapeutic proteins in high levels. However, the production of recombinant proteins often results in the accumulation of insoluble aggregates (inclusion bodies, IB) in bacterial cytoplasm, which is a serious problem for biotechnology because it decreased the yield of bioactive proteins. The application of high pressure is a mild technique for dissociation of protein aggregates which enables maintenance of secondary and tertiary structures, and can be useful for proteins refolding to increase yields of native proteins. In this project, we will develop models for using high pressure to promote protein refolding from aggregates to increase yields of natively active proteins. To this end, we will conduct studies of protein stability and characterization of intermediate protein states in the unfolding/folding pathways of human growth hormone (hGH) and cruzain. We will optimize the conditions of pressure, temperature and buffer for the acquisition of high levels of active native state using high hydrostatic pressure for each of the monomeric proteins Bcp, TrxA, TsnC and Ybbn of X. fastidiosa, PilB of X. axonopodis pv citri, SM29 from S. mansoni and GFP and the oligomeric proteins CTB and fiber knob of adenovirus. We will also study the cultivation conditions to optimize the production of easily dissociable IB of the recombinant proteins. (AU)

Scientific publications (4)
(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)
LEMKE, LAURA SIMONI; CHURA-CHAMBI, ROSA MARIA; RODRIGUES, DANIELLA; ROSA CUSSIOL, JOSE RENATO; MALAVASI, NATALIA VALLEJO; PIRES ALEGRIA, THIAGO GERONIMO; SOARES NETTO, LUIS EDUARDO; MORGANTI, LIGIA. Investigation on solubilization protocols in the refolding of the thioredoxin TsnC from Xylella fastidiosa by high hydrostatic pressure approach. Protein Expression and Purification, v. 106, p. 72-77, FEB 2015. Web of Science Citations: 2.
RODRIGUES, D.; FARINHA-ARCIERI, L. E.; VENTURA, A. M.; CHURA-CHAMBI, R. M.; MALAVASI, N. V.; LEMKE, L. S.; GUIMARAES, J. S.; HO, P. L.; MORGANTI, L. Effect of pressure on refolding of recombinant pentameric cholera toxin B. Journal of Biotechnology, v. 173, p. 98-105, MAR 10 2014. Web of Science Citations: 6.
MALAVASI, N. V.; CORDEIRO, Y.; RODRIGUES, D.; CHURA-CHAMBI, R. M.; LEMKE, L. S.; MORGANTI, L. The effect of temperature on protein refolding at high pressure: Enhanced green fluorescent protein as a model. Process Biochemistry, v. 49, n. 1, p. 54-60, JAN 2014. Web of Science Citations: 0.
CHURA-CHAMBI, R. M.; CORDEIRO, Y.; MALAVASI, N. V.; LEMKE, L. S.; RODRIGUES, D.; MORGANTI, L. An analysis of the factors that affect the dissociation of inclusion bodies and the refolding of endostatin under high pressure. Process Biochemistry, v. 48, n. 2, p. 250-259, FEB 2013. Web of Science Citations: 11.

Please report errors in scientific publications list by writing to: cdi@fapesp.br.