Fig 1.
Purified recombinant SEAP examined by SDS-PAGE analysis.
SEAP was produced in HEK 293 cells and purified from medium containing 10% serum. Lanes 1–3, SEAP-6×His purified on Ni-NTA magnetic agarose beads; lane 4, molecular weight standards; lanes 5–7, SEAP-HA purified on magnetic beads with immobilized anti-HA antibodies. The method of elution is shown at the top of each lane.
Fig 2.
Relative secretion level of SEAP from HEK293T cells transfected with modified psiTEST vector.
The amount of phosphatase was determined using QUANTI-Blue reagent, as described in Materials and Methods. All experiments were performed in triplicate. All values were expressed as means ± SD. The calculated secretion rate for SEAP-6×His with additional N-glycosylation sites introduced at positions 150 and 278 was approximately 12 μg per 1 ml of the medium. The inset at the top presents SDS-PAGE of SEAP-6×His purified on magnetic Ni-NTA beads from serum-free media collected from HEK293T cells, transfected with vector coding wild-type enzyme (lane 1), enzyme with additional glycosylation site at position 278 (lane 2), and double mutant SEAP with glycosylation sites introduced at positions 150 and 278 (lane 3), respectively. 1 μg of the protein was applied into each lane; lane 4 –molecular weight standards. Statistical significance was assigned to p-value < 0.05. ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Fig 3.
Comparison of three variants of recombined and enzymatically active SEAP.
The enzyme was bound on magnetic beads and released with imidazole. Specific activity was calculated from end-point reactions of p-nitrophenyl phosphate as substrate (see Materials and Methods for details). Statistical significance was assigned to p-value < 0.05. ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Table 1.
Constructs tested for phosphatase secretion and activity.
Fig 4.
Analysis of abundance of high-mannose structures.
N-glycan profiles of HEK293T total lysate and SEAP-6×His with new glycosylation sites at 150 and 278 positions, purified on Ni-NTA beads from standard medium, are shown. Top panels–before mannosidase treatment; bottom panels–after mannosidase digestion. Peak on the left shows the product of digestion of all high-mannose type structures (blue, solid square–N-acetylglucosamine, green, solid circle–mannose).
Fig 5.
N-glycosylation profiles of modified SEAP, purified from media of HEK293T, HepG2 and CHO cell cultures.
Fig 6.
Schematic description of proposed conditions for N-glycan isolation from recombined SEAP.