Cloning, sequencing and expression of a 5-lipoxygenase from Syrian hamster embryo fibroblasts

Abstract

The hamster ortholog of human and rat 5-lipoxygenase (5-LO) was cloned from a Syrian hamster embryo (SHE) cell line. A combination of polymerase chain reaction (PCR) and 5' and 3' RACE (rapid amplification of cDNA ends) was used to isolate the complete cDNA for this gene. The cDNA sequence demonstrates the extreme sequence conservation found in this gene family, with a deduced amino acid sequence 95% identical to the rat 5-LO, and 90% identical to the human enzyme. The hamster 5-LO was expressed in E. coli. The expressed protein was detected by an antibody to human 5-LO, and had an apparent molecular weight of 75-80 kD. The products of the action of this enzyme on arachidonic acid are 5-HETE and the diHETEs resulting from the breakdown of LTA4, in a pattern similar to that produced by the recombinant human 5-LO. No oxidation of linoleic acid by this enzyme was detected.