Expression, purification and preliminary structural studies of T. cruzi mRNA maduration proteins

CARMONA N, GÓMEZ BARROSO JA, BERCOVICH N, VAZQUEZ M, AGUILAR CF

San Luis

Congreso; XXVII REUNION CIENTIFICA ANUAL DE LA SOCIEDAD DE BIOLOGIA DE CUYO; 2009

Sociedad de Biología de Cuyo

<!-- /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman"; mso-ansi-language:EN-GB;} a:link, span.MsoHyperlink {color:blue; text-decoration:underline; text-underline:single;} a:visited, span.MsoHyperlinkFollowed {color:purple; text-decoration:underline; text-underline:single;} @page Section1 {size:612.0pt 792.0pt; margin:70.85pt 3.0cm 70.85pt 3.0cm; mso-header-margin:36.0pt; mso-footer-margin:36.0pt; mso-paper-source:0;} div.Section1 {page:Section1;} --> Chagas’ disease is caused by the flagellate protozoan Trypanosoma cruzi. Current drug treatments are generally unsatisfactory. Available medications are highly toxic and often ineffective, particularly those used to treat the chronic stage of the disease. The objective of our work is the resolution by X-ray crystallography of the three-dimensional structure of proteins which are vital for T. cruzi. This information may provide new drug targets for the treatment of the disease. The T. cruzi proteins studied in this work are: TcFIP1-like (factor interacting with Pap1) and TcCPSF30 (cleavaje and polyadenylation factor 30), both involved in mRNA cleavaje and polyadenylation. They were expressed in Escherichia coli fused to a N-terminal His-tag. The protein purification was optimized using different methods. TcCPSF30 was correctly refolded in vitro. This refolded protein was used for subsequent native PAGE, transverse urea-gradient, and other conformational assays. Modelling studies of these two proteins using homologous part of different proteins have given important structural clues.