Figure 1

Construction, N- glycosylation and expression of hPGHSs. A, the positions of the N-glycosylation sites of the chimera and hPGHS isoforms. The dashed lines connect N- glycosylation sites that are conserved between isoforms. Asparagines, which are not glycosylated in native hPGHSs, are depicted with grey characters. N580 is designated by dark grey characters as about 50% of the native PGHS-2 is not glycosylated at this site. The transition from hPGHS-2 to hPGHS-1 to form the chimera is shown as well. B, Western blot analyses of the microsomes of P. pastoris cells expressing the non-optimised hPGHS-1 (1) and the codon optimised hPGHS-1 (2). hPGHS-1 was expressed with the native signal sequence and the optimised sequence contained an affinity tag. C, Western blot analysis of the purified hPGHSs subjected to N-glycosylation analysis: hPGHS-1 (lane 1), chimera (lane 2) and hPGHS-2 (lane 3). The calculated molecular weights of the non-glycosylated proteins were 67.2, 66.3 and 68.8 kDa, respectively, assuming that the signal peptide was cleaved and taking into account the double STEL and polyhistidine tag.