Clonagem e expressão de irisina canina em Escherichia coli

Abstract

Introduction: Irisin is an endogenously produced protein upon continuous physical stimulation. It is a product of the cleavage of fibronectin type III transmembrane protein containing domain 5 (FNDC5). In addition to the signal peptide and the C-terminal portion, FNDC5 has a transmembrane region and the irisin domain. Physical exercise has a direct action on protein synthesis, since it activates the expression of the PGC1-α gene that induces the synthesis of the FNDC5 protein with consequent release of irisin. In adipose tissue, it acts positively on the UCP1 molecule, leading to increased mitochondrial density, high oxygen consumption and a change in the phenotypic characteristics of white adipocytes to beige. These characteristics are associated with weight loss, improved insulin resistance and regression of metabolic syndrome. In the central nervous system it promotes the survival, maintenance and function of neural cells. In dogs, as in humans, obesity is a risk factor for the development of metabolic dysfunctions such as diabetes mellitus, altered lipid profiles and hypertension. Canine cognitive dysfunction affects about 60% of older dogs, and is related to the deposition of beta-amyloid molecules. Aiming at the future development of a drug to treat these canine dysfunctions, this project aims to clone and express the coding sequence of canine irisin in Escherichia coli. Material and Methods: The gene sequence of the protein was selected from the UniProt database. The sequence alignment in order to analyze the similarity between humans and dogs was done using the Blast-p program. Once the sequence was selected, the gene was designed and ordered for chemical synthesis with E.coli preferred codons and cloned into the pUC-18 vector. The irisin sequence carrying Ndel and BamHI restriction enzyme sites at the ends was subcloned into the sites of the same enzymes in the expression vector pDMU01, resulting in the recombinant plasmid pTI01. For transformation and gene expression pTl01 was introduced by electroporation into E. coli strain DH5αF'Iq. A recombinant colony was inoculated into 50 mL of Luria Bertani medium containing ampicillin 100 ug/mL o and grown at 37 oC, 150 rpm rotation for 32 h. When the culture reached 0.5 OD600/mL expression was induced by adding IPTG to the final concentration of 1 mM. During growth aliquots were collected at specific time slots for absorbance measurement and analysis of recombinant protein by SDS-PAGE electrophoresis. Results and discussion: Digestion of the pUC18-IRISINA vector containing the expression gene as well as the pDMU01 expression vector with the restriction enzymes Ndel and BamHI was efficiently performed. The binding of the gene to the pDMU01 plasmid was confirmed by enzymatic digestion using the respective enzymes, and it was possible to observe in agarose gel the release of a 432pb fragment of PTI01 referring to the irisin coding region. Gene expression was confirmed by SDS-PAGE gel where a band of approximately 17kDa corresponding to the molecular mass of canine irisin was observed.