Production of bovine recombinant leptin in Pichia pastoris yeast and evaluation of the biological activity

• Grant number: 11/02206-0
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: June 01, 2011
• End date: January 31, 2014
• Field of knowledge: Agronomical Sciences - Animal Husbandry - Animal Production
• Principal Investigator: Luis Felipe Prada e Silva
• Grantee: Marina Vieira de Carvalho
• Host Institution: Faculdade de Medicina Veterinária e Zootecnia (FMVZ). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

Nutrition has a clear effect on the reproductive development of cattle, with leptin being a candidate for a likely link between nutrition and reproduction by signaling body fatness to the hypothalamus and therefore allowing puberty to occur. Our objective with this study is to clone the coding region of the bovine leptin gene and to produce the recombinant bovine leptin in Pichia pastoris yeast, and finally to evaluate its biological activity. The coding region of bovine leptin will be obtained by PCR amplification of the transcript cDNA from RNA extracted from adipose tissue of Nellore heifers. The gene will be cloned in expression vector and then inserted by electroporation into Escherichia coli TOP10TM bacteria. After selection of the colonies, the plasmids will be isolated and sequenced to confirm that the insert is aligned with the signaling sequence of secretion. Strains of P. pastoris will be transformed by electroporation for secretion of leptin into culture media, with the leptin being then purified by three phases of chromatography, dialyzed and lyophilized. An in vitro assay will then be performed with BAF/3 1442-C14 cells, transfected with the human leptin receptor, to evaluate its proliferation in the presence of the recombinant leptin produced. (AU)

Academic Publications

(References retrieved automatically from State of São Paulo Research Institutions)

CARVALHO, Marina Vieira de. Production of recombinant bovine leptin in Pichia pastoris yeasts and evaluation of the biological activity. 2014. Doctoral Thesis - Universidade de São Paulo (USP). Faculdade de Medicina Veterinária e Zootecnia (FMVZ/SBD) Pirassununga.