Production of recombinant SphK1 for in vitro sphingosine kinase activity assays

ALVAREZ SE; CARMONA N; MALDONADO R; MASCOTTI ML

Congreso; XXXV Reunión Científica Anual de la Sociedad 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. coli cells 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). SDS-PAGE and Western blot expression was tested in different E. coli strains: 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.