Y varieties of tumor cells, and are involved in tumor progression and aggressiveness (ten,11). Also, the expression of COX2 was observed to be induced in cancer cells during anticancer chemoradiotherapies, resulting in drug resistance (1113). Thus, the inhibition of COX2 may perhaps supply a very considerable therapy which may possibly benefit a big proportion in the patient population (10). While broad spectrum COX2inhibiting nonsteroidal antiinflammatory drugs (NSAIDs), and COX2specific inhibitors have been successfully established (11,12), both of these are identified to bring about sideeffects, including myocardial infarction (11). For that reason, there nevertheless remains an urgent want to create antiCOX2 therapies with decreased or no sideeffects. The preparation of COX2 protein could be the initial step for the development of COX2 inhibitors. A eukaryotic hemecontaining and membranebound protein, COX2 is expressed at a ratherCorrespondence to: Professor Guiying Li, Important Laboratory forMolecular Enzymology and Engineering of the Ministry of Education, College of Life Sciences, Jilin University, 2699 Qianjin Street, Changchun, Jilin 130012, P.R. China Email: [email protected]: COXs, cyclooxygenases; PGs, prostaglandins; AA,arachidonic acid; PGE2, prostaglandin E2; trCOX2, truncated human COXKey words: human cyclooxygenase, prokaryotic expression, inclusionbodies, purification, laptop or computer simulationLIAO et al: PROKARYOTIC EXPRESSION AND PURIFICATION OF HUMAN COXlow level in native hosts. Heterologous expression may be the only efficient approach with which to acquire a sizable level of human COX2 protein. Normally, by far the most frequently utilized heterologous expression systems incorporate prokaryotic, yeast, plantbased, insect/baculovirus and mammalian expression systems, too as expression in eukaryotic organisms (14,15). Together with the progression of COX2 structural studies (36), the insect/baculovirus expression technique has turn into by far the most widespread approach for acquiring higher high-quality functional products (1619). Having said that, numerous limitations in the insect/baculovirus method, such as a comparatively high cost, methodological challenges and relatively low (��)-Alliin Inhibitor yields obtained working with this method, limit its use for largescale fermentation and more widespread application. Furthermore, protein synthesis rates are typically a lot more rapidly in prokaryotes than in eukaryotes (20). Therefore, bacterial hosts are preferred, as a consequence of their fast development rate, their capacity for continuous fermentation, highlevel postinduction target protein expression and also a comparatively low price (14,2026). Even so, to date, and no less than towards the greatest of our understanding, restricted study has been carried out to characterize and purify human COX2 expressed in prokaryotic cells (27). Within this study, a truncated form of human COX2, containing 257 residues in the Cterminus was cloned, and it exhibited highlevel heterologous expression in Escherichia coli (E. coli) BL21(DE3) cells using the pET28b() expression vector method. In addition, the antigenicity and also the COX activity of truncated human COX2 (trCOX2) merchandise had been validated and these outcomes demonstrate the reliability of this technique to get functional COX2 merchandise from a prokaryotic expression system. Supplies and Ciprofloxacin (hydrochloride monohydrate) Bacterial solutions Materials. BamHI, HindIII and T4 DNA ligase have been all purchased from Takara Biotechnology Co., Ltd. (Dalian, China). A Ni2NTA Superflow Cartridge was purchased from Qiagen (Valencia, CA, USA). PD10 desalting columns had been obtained from Amersham Pharmacia Biotech, Inc. (Pi.