Summary: | 碩士 === 輔仁大學 === 生命科學系碩士班 === 96 === The prostaglandins are lipid mediators, which are made by the bifunctional enzyme, Cyclooxygenase(COX), containing both cyclooxygenase and peroxidase activities. Prostaglandins are involved in physiological functions such as protection of the stomach mucosa, aggregation of platelets and regulation of kidney function...etc. They also have pathological functions such as induction of inflammation, fever and pain...etc. There are two isoforms of cyclooxygenase: Cyclooxygenase-1(COX-1)and Cyclooxygenase-2(COX-2). The COX-1 is a housekeeping or constitutive enzyme making prostaglandins which are important for maintaining physiological functions. In this study, a recombinant human COX-1(hCOX-1; human prostaglandin H synthase 1, PGHS1)was expressed in yeast, K. lactis, and 293T mammalian cells, but not in NT-2 cells. In the yeast system, the full-length cDNA for hCOX-1 was placed under the control of the LAC4 promoter and was integrated into the yeast genome by homologous recombination. The hCOX-1 enzyme was extracted from the transformed yeast by a solution containing 0.2% of Triton X-100, and the efficacy of hCOX-1 enzyme extraction was over 40% as estimated by Western blotting. In the mammalian cell system, the full-length cDNA for hCOX-1 was under the control of CMV ( cytomegalovirus) promoter from the mammalian expression vector, pcDNA3.1, and was transiently transfected into both NT-2 and 293T cells. The recombinant hCOX-1 enzyme expressed in both yeast and mammalian systems contains six-histidine tag (His-tag) at the carboxyl terminus, and was identified by Western blotting using both anti-COX-1 and anti-His antibodies. Although no recombinant hCOX-1 proteins were identified in NT-2 cells, and only small amounts of the recombinant hCOX-1 were detected in the transfected 293T cells, a larger quantity of recombinant proteins was found in the yeast expression system. Previously, it was reported that hCOX-1 successfully expressed in both the BVES (baculovirus expression system)and in the VV (vaccinia vector transfected)COS-7 cells. However, both processes were rather complicated and involving the use of viral vectors which carry potential hazards of environmental contamination. The data presented here demonstrate that it is possible to produce hCOX-1 enzyme for biochemical, biophysical, and pharmacological investigations using both the yeast K. lactis and 293T mammalian cell expression systems circumventing the risk of possible viral contamination. The low expression of recombinant hCOX-1 in transfected 293T cells is possibly caused by cellular auto-regulation mechanism to avoid toxicity of prostaglandin over-production. Further investigation will warrant the production of recombinant hCOX-1 in the mammalian cells via simple transfection.
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