PRODUCTION OF RECOMBINANT SPHK1 FOR IN VITRO SPHINGOSINE KINASE ACTIVITY ASSAYS

MALDONADO, R; CARMONA, N; ALVAREZ, S; MASCOTTI, ML

Congreso; XXXVReunión Científica Anual de laSociedad de Biología de Cuyo; 2017

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid modulating numerous cellular processes as  cancer,  proliferation,  migration and  apoptosis  among  others.  This molecule  is  produced  by  the so-called  sphingosine  kinases  (SphKs)  which  carry  out  the  phosphorylation  of  sphingosine  at expenses  of  consuming  ATP. There are two  isoforms  of  SphKs  in humans,  SphK1  and  SphK2.  It has been reported that SphK1 is overexpressed in different kind of cancers, including solid tumors and  leukemia.  Also,  the  incremented  expression  of this  enzyme  has  been  related  to  resistance  to chemotherapy.  Therefore,  finding  inhibitors  of  SphK1  in humans may  represent  a novel  target  in cancer research. In this context, the aim of our work was to produce recombinant SphK1 in E. colicells and purify it to be further employed in screening assays searching for effective inhibitors. The coding  sequence  of  Sphk1  was  kindly  provided  by  the  group  of  Dr.  Spiegel  (USA)  in  a  pET16b expression vector. Restriction analysis and Sanger sequencing were carried out in order to confirm the  identity  of  the  catalytic  domain  of  SphK1  (1152  bp).  SphK1  expression  was  tested  by  SDS-PAGE and Western blot in different E. colistrains: BL21, BL21pLysE and Rosetta, at 4 different induction temperatures (18, 24, 30 and 37°C), various IPTG concentrations and induction times. The results  suggest  that  best  expression  level  is  achieved  in  BL21  strain,  at  24  and  30°C  after  6  h incubation. Currently, we are working on expression conditions to get SphK1 in soluble form to be further purified by affinity chromatography and tested.