Cloning of the gene TcPEPCK and expression of phoenolpiruvate carboxikinase protein from Trypaonosoma cruzi in E.coli

Abstract

Chagas disease, caused by the protozoan Trypanosoma cruzi, affects approximately 20 million people currently and it is considered a neglected disease. It is most commonly found in Central and South America. The vector of transmission is an insect commonly known as blood-sucking assassin bugs that during the night or during the day in dark, get out of their hiding places, usually cracks in walls, mattresses, straw roofs or clay, to suck the blood of the exposed parts of a mammalian host. The transmission of the parasite occurs through the feces of the insect, and once infected, the individual goes through two stages of the disease: acute and chronic phase. In the final stage of the disease, heart failure and gastrointestinal inflammation results 10% in fatalities. The fact that the drugs used to combat Chagas disease are not effective and cause serious side effects (such as anorexia, weight loss, hyperexcitability and bone marrow depression, dizziness, headache and abdominal pain) motivates the development of new drugs that are effective and less toxic. The development of new drugs requires the full knowledge of the life cycle and metabolic mechanisms of T. cruzi. One way to develop new drugs against Chagas disease is to exploit the fact that the infective form of T. cruzi depend solely on glycolysis as an energy source. Therefore, compounds that block glycolysis may be used as drugs to fight the disease. An approach to the search for compounds with potential inhibitory capacity of enzymes is the scanning (screening) of combinatorial libraries and libraries of natural compounds. The enzyme phosphoenolpyruvate carboxykinase (PEPCK) is an enzyme in the glycolytic pathway that catalyzes reversibly the carboxylation of phosphoenolpyruvate and its dephosphorylation, forming oxaloacetate (OAA). In previous studies of T. cruzi PEPCK enzyme was expressed and had its three-dimensional structure determined (Trapani et al., 2001). Because the enzyme is expressed without fusion, it requires five purification steps, resulting in low yields. Moreover, some of the resins once used were discontinued, which prevents the use of previously developed protocol. The search for inhibitors of T. cruzi PEPCK is one of the projects entered into the National Institute of Structural and Medicinal Chemistry and Biotechnology of Infectious Diseases (INBEQMeDI-INCT/CNPq), which is coordinated by Prof. Dr. Glaucius Oliva, from Institute of Physics, São Carlos. For the test of the enzyme PEPCK against possible inhibitors is necessary that it be expressed in soluble form, purified in high purity and to establish a methodology for the screening of compounds. Thus, in this project we propose cloning the Tcpepck gene into a vector allowing the expression of a protein fused with a peptide histidine and expression of the enzyme PEPCK. Once the pure enzyme we will be studying the kinetic parameters of the enzyme for further use in the search for natural or synthetic inhibitors. The search for inhibitors of PEPCK will be held in collaboration with Profs. Dr. Arlene G. Correa, Paulo Cezar Vieira, from Department of Chemistry, UFSCar, which have libraries of such compounds.