Cloning, expression and purification of SIT4, a Ser/Thr phosphatase from Saccharomyces cerevisiae
Leandro José de Assis, Willy Jablonka and Monica Montero-Lomeli
Laboratório de Biologia Molecular e Bioquímica de Leveduras - Instituto de Bioquímica Médica - Programa de Biologia Molecular e Biotecnologia - Universidade Federal do Rio de Janeiro - UFRJ
The SIT4 gene from yeast Saccharomyces cerevisiae encodes a PP2A-related protein Ser/Thr phosphatase which regulates other proteins in important cellular processes like cell cycle control, glycogen metabolism and cell wall integrity. SIT4 participates in a conserved pathway, the TOR pathway (target of rapamycin), which connects nutrient availability to cell growth and is activated in mammalian cancer cells. In humans, PP6 is a SIT4 homolog involved with spliceosomal small nuclear ribonucleoproteins in lymphocytes and related to the interleukin-2 pathway in T cells. Despite knowledge of some functions, the Sit4p structure, activity in vitro and targets in vivo are not known yet. In this work we cloned and expressed SIT4 in E. coli M15 with N-terminal his-tag. We expressed SIT4 in great quantity in E. coli M15 but verify its direction to inclusion bodies. For Sit4p purification we dissolved the inclusion bodies in 8 M urea followed by gel filtration chromatography and tried to renature it by dialysis. We also analyzed Sit4p structure (311 residues) in silico and recognized a globular structure with two plausible disulfide bonds between aminoacids C263/C260 and C139/C24 or C139/C126. The in silico structure also shows a Ser/Thr phosphatase conserved folding: two parallel b-sheets in a b-sandwich surrounded by a-helixes. This data is important for the isolation and for studying the kinetic characteristics of this important phosphatase which can be used as a target in the treatment of cancer.
This work was supported by CNPq and FAPERJ.
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