Expression Of The RNA Binding Proteins CIRBP And SFRS9 For Functional And Structural Studies
Kuniyoshi, T.M.1, Nery, F.C. 1, Camargo, M.E.R. 1, Kobarg, J. 1
1LNLS – Laboratório Nacional de Luz Síncrotron/CeBiME- Centro de Biologia Molecular Estrutural – Campinas-SP-Brasil- jkobarg@lnls.br
The protein Ki-1/57 was first identified by the cross reaction of the CD30 monoclonal antibody Ki-1 in Hodgkin lymphoma cell. The expression of Ki-1/57 in diverse cancer cells and its phosphorylation in blood leukocytes only after mitogenic activation, suggested a role of this protein in the cellular activation and possibly in cell signaling. Furthermore, this protein of 57 KDa, is associated with a serine/threonine protein kinase activity in cancer cells (1) and electron microscopic analysis showed that the Ki-1/57 antigen is located in the cytoplasm, at the nuclear pores and in the nucleus. To obtain clues for the functional context of Ki-1/57 protein, our group explored the yeast two-hybrid system to identify interacting proteins. Our analyses resulted in the identification of several proteins, including: Chromo-Helicase-DNA-binding domain protein-3 (CHD3) (2), RACK-1 (Receptor of Activated Kinase 1) (3) and two small proteins that contain RNA binding domains: CIRBP (Cold Induced RNA Binding Protein) and SFRS9 (splice factor Arg/Ser rich 9). In the present work we expressed CIRBP and SFRS9 for both structural and functional studies. Firstly, the cDNAs encoding full-length CIRBP and SFRS9 were sub-cloned into bacterial expression vectors. GST-CIRBP and 6xHis-CIRBP fusion protein expression and purification were optimized. Since SFRS9 protein did not express in both bacterial fusion systems, we opted to employ the baculo virus expression system and were able to obtain soluble 6xHis-SFRS9 protein. We used the recombinant proteins to performed pull down assays and were able to confirm the interaction of GST-CIRBP with full-length and N- and C-terminal Ki-1/57 deletion constructions. In the moment we are performing detailed mapping studies of the interaction between Ki-1/57 and SFRS9 and are using both recombinant proteins in spectroscopic studies and crystallization trials.
Supported by: FAPESP, LNLS, CNPq
References:
(1)Kobarg et al., 1997 Exp. Clin. Immunogen. 14: 273-80.
(2)Lemos et al., 2003, FEBS Lett. 533: 14-20.
(3)Nery et al., 2004, J Biol Chem 279: 11444-55.
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