Total product weight from 1 liter culture medium. trCOX2, truncated human cyclooxygenase2; E. coli, Escherichia coli.Monomethyl MedChemExpress Figure four. Analysis of truncated human cyclooxygenase2 (trCOX2) expression in Escherichia coli (E. coli) BL21(DE3) by 12 SDSPAGE. Lane 1, protein molecular weight regular; lane two, cell lysate of E. coli BL21(DE3); lane 3, cell lysate of pET28b/BL21(DE3); lane four, cell lysate of pET28btrCOX2/BL21(DE3) without having induction; lanes 59, cell lysate of pET28btrCOX2/BL21(DE3) induced by isopropyl D1thiogalactopyranoside (IPTG) for 2, three, four, 6 and eight h, respectively.of trCOX2. The fulllength in the fusion protein with Histags, trCOX2, was 305 amino acids (34.four kDa). Expression and purification of trCOX2. To obtain human trCOX2 protein, competent E. coli BL21(DE3) cells have been transformed with pET28btrCOX2 to prepare E. coli trCOX2/BL21(DE3) that could express human trCOX2. We identified that the expression degree of the trCOX2 protein was extremely higher soon after IPTG induction, as detected by SDSPAGE (Fig. four). Moreover, the expression of target proteins reached the highest level (up to 31 in the total E. coli protein) at 4 h right after IPTG induction (Fig. 4), however they were expressed as inclusion bodies as they were located in the pellets of cell lysates (Fig. 5). To be able to purify trCOX2, the pellets containing the inclusion bodies had been very first washed with Triton X100 and 2 M urea to receive crude inclusion bodies, which were then solubilized applying ureadenaturation. The soluble inclusion physique proteins with Histags were then subjected to affinity purification. SDSPAGE analysis with the eluted fractions revealed that a single band of approximately 34 kDa was detected (Fig. 5). The purity on the productsexpressed inside a prokaryotic expression system, we cloned trCOX2 and constructed a prokaryotic expression plasmid. As shown in Fig. 3, the 771 bp PCR solution encoding the Cterminal segment of human COX2 (like 257 amino acid residents) was cloned successfully and inserted in to the prokaryotic expression vector pET28b(). Constructive recombinant Colistin methanesulfonate (sodium salt) site plasmids had been confirmed with digestion working with BamHI and HindIII enzymes (Fig. 3). The sequencing benefits provided additional evidence of productive construction on the recombinant pET28btrCOX2 expression plasmid and confirmed the presence of two 6xHistags, situated at each the N and CterminusLIAO et al: PROKARYOTIC EXPRESSION AND PURIFICATION OF HUMAN COXFigure 6. Identification of recombinant truncated human cyclooxygenase2 (trCOX2) protein by western blot and ELISA assays. (A) Western blot evaluation of trCOX2 with antiHistag antibody. Samples have been loaded as follows: lane 1, pET28btrCOX2/BL21(DE3) devoid of induction; lane two, pET28btrCOX2/BL21(DE3) induced by isopropyl D1thiogalactopyranoside (IPTG) for 4 h; and lane three, recombinant trCOX2 protein. (B) Western blot evaluation of trCOX2 with antiCOX2 antibody. Samples have been loaded as follows: lane 1, BL21(DE3); lane two, pET28btrCOX2/BL21(DE3) without having induction; lane 3, pET28btrCOX2/BL21(DE3) induced by IPTG for four h; and lane four, recombinant trCOX2 protein. (C) ELISA assay of trCOX2 with antiCOX1 and COX2 antibodies.Figure 7. Cyclooxygenase (COX) assay of truncated human COX2 (trCOX2). COX activity measured by relative oxygen concentration. A reduce final oxygen concentration indicates higher oxygen consumption and larger COX activity.In order to examine the antigenicity and binding activity of ready trCOX2 to antiCOX2 or antiCOX1 antibody, an ELISA assay was performed. As shown in.