Figure 1.
Design of the different NA expression constructs.
The figure shows the constitution and partial sequence of the different NA expression constructs used to express secreted NA: Recombinant Hokkaido H1N1 NA with an artificial stalk based on the Yeast transcription factor GCN-pLI (A) or the Tetrabrachion tetramerization domain of Staphylothermus marinus (B); The third construct is based on the Tetrabrachion stalk fused to the NA head sequence of pN1/2009 NA. It contains a Thrombin cleavage site C-terminal of the FLAG allowing the efficient cleavage of the tag. Constructs A to C use the Melittin signal peptide (MSP) to drive secretion of the respective NA. Figure 1D shows the sequence of the construct used to express Hokkaido H1N1 NA in mammalian cells. This construct uses the mouse Interleukin 3 (IL3) secretion signal to drive protein secretion.
Figure 2.
Expression and purification of recombinant NA in insect cells.
The expression and secretion of GCN-pLI and TB-based soluble Hokkaido NA was monitored by anti-FLAG WB (2A, B) and by NA activity assays (2C). The gel filtration chromatogram for both NA constructs is shown in 2D. 2E shows the processed insect cell media before and after anti-FLAG affinity purification in a Coomassie stained gel (2E left panel) and corresponding WB (2E right panel). Figure 2F shows the FLAG- and Lectin/IEX-purified pN1/2009 NA loaded onto an SDS-PAGE and stained by Coomassie (2F left panel) or detected by anti-FLAG WB (2F right panel). The amino acid sequence obtained by N-terminal sequencing of both enzymes is shown in 2G.
Figure 3.
Identification of FLAG-tag tyrosine-sulfation.
Lectin purified TB-Hokkaido NA was incubated with different PTM-cleaving enzymes. The anti-FLAG WB is shown in Fig. 3A. Addition of sulfatase made the FLAG tag accessible to the anti-FLAG mAb. Desulfation kinetics of Lectin/IEX-purified pN1/2009 NA using sulfatase type VIII (Abalone entrails) are shown in 3B. Activity of all 4 sulfatases tested was efficiently inhibited by the addition of vanadate-phenyl-Ester (3C). Fig. 3D shows the negative linear mode MS of the sulfated control peptide CCK-8 at 1143.5 Da and its non-sulfated form at 1063.3 Da (−80 Da). The Thrombin cleaved FLAG tag was detected in its sulfated and non-sulfated form at a molecular weight of 1690.9 and 1611.1 Da (−80 Da), respectively. Fig. 3E shows the same sample measured in positive linear mode. Only the non-sulfated peptides (−80 Da) were detected with an additional weight due to protonation (+1 Da). Fig. 3F shows the WB of FLAG- and Lectin-purified NA (pN1/2009) detected with different anti-FLAG antibodies as well as the Coomassie stained gel of the same samples.
Table 1.
Theoretical and experimental peptide masses.
Table 2.
Anti-FLAG antibodies used to detect FLAG-tagged NA.
Figure 4.
Expression and purification of recombinant NA in mammalian cells.
Human TB-NA (Hokkaido) fused to the Tetrabrachion stalk (Fig. 1B) was cloned into the pApex-3 vector and expressed in HEK293T cells. Media, flow-through, and purified NA were loaded onto SDS-PAGE and detected by anti-FLAG WB (left panel) or stained by Coomassie (right panel). Secreted FLAG-reactive NA was detected by anti-FLAG WB. Based on the anti-FLAG WB the column flow-through did not contain any residual FLAG reactive NA.