Detection of soluble HAs in cell culture supernatants and cell lysates

Figure 26. Expression and detection of pCG1 in HEK-293T cells. HEK-293T cells were transfected with the empty vector as a negative control and fixed 24 h post transfection. An -human IgG FITC antibody was used for detection. The images were taken with a confocal laser scanning microscope with 63x magnification (left panel). The right panel shows a close-up view. Scale bar left panel: 50 µm;

scale bar right panel: 25 µm.

4.3.2 Detection of soluble HAs in cell culture supernatants and cell lysates

After having confirmed that the different soluble hemagglutinins were expressed in HEK-293T cells, the secretion of the proteins into the cell culture supernatant was analyzed by Western Blot.

For this purpose, the supernatants of transfected HEK-293T cells were harvested 24 h post transfection (3.1.5.2). The soluble proteins were separated under reducing and non-reducing conditions (3.6.3), separated by electrophoresis, transferred to nitrocellulose membrane and detected by chemiluminescence (3.6.4).

For the confirmation of the presence of soluble proteins in the cell culture supernatant, on the one hand the anti-H1 antibody from the previous experiments was used, and on the other hand an antibody which recognizes the human Fc-tag (anti-human-PO) was used. As a negative control, the supernatant of cells transfected with the empty vector pCG1 was used.

pCG1

Figure 27. Detection of the soluble hemagglutinins in the supernatant of transfected HEK-293T cells. Supernatants of cells expressing either of the soluble hemagglutinins were analyzed by SDS-Page under non-reducing (A) or reducing (B) conditions. Following transfer to nitrocellulose, hemagglutinins were visualized by immunostaining using an H1antibody and HRP conjugated -mouse antibody.

Figure 28. Detection of the soluble hemagglutinins in the cell lysates of transfected HEK-293T cells. Cell lysates of cells expressing either of the soluble hemagglutinins were analyzed by SDS-Page under non-reducing (C) or reducing (D) conditions. Following transfer to nitrocellulose, hemagglutinins were visualized by immunostaining using an H1antibody and HRP conjugated -mouse antibody.

A B

C D

Figure 29. Detection of the soluble hemagglutinins carrying the Fc-tag in the supernatant of transfected HEK-293T cells. E) Supernatants of 1918_Fc, 2009_Fc and Fc-ATG under non-reducing (left) and reducing conditions (right) detected with the -human-PO antibody. Black arrow: trimeric form of the soluble HAs.

Figure 27, 28 and 29 show the detection of the soluble hemagglutinins in the cell culture supernatants and cell lysates of transfected HEK-293T cells. The different constructs, except for the 1918_T6his construct, could be detected in the supernatants of transfected HEK-293T cells with the anti-H1 antibody (figure 27).

Furthermore, the Fc-constructs in the supernatants were also detectable with the anti-human-PO antibody (figure 29). In the cell lysates, it was possible to identify all soluble hemagglutinins, also the 1918_T6his, by using the anti-H1 antibody (figure 28). The transfection of HEK-293T cells with the Fc-ATG plasmid resulted in the secretion of the Fc into the supernatant. A strong band of about 35 kDa represents the monomeric form of the Fc (figure 29).

The detection of the soluble proteins in the supernatants showed that under non-reducing conditions all proteins are detectable only as high molecular bands with a size of 300 kDa and more (figure 27.A). As the monomeric form of the 1918 hemagglutinin has a size of approximately 60 kDa when glycosylated, the bands detected on the Western Blot represent multimeric forms of the soluble proteins. An exception are the 1918_sol and the 1918_Fc construct, which are present also in a trimeric form at the level of 270 kDa, as a monomer has a size of about 90 kDa

E

(figure 27.A and figure 29, black arrow). Analysis of the supernatants under reducing conditions shows that only in the supernatants of 1918_sol and 1918_Fc there are multimeric forms of the proteins detectable (black arrow, figure 27.B). In the various supernatants without Fc-tag, only bands with a size of around 100 to 115 kDa are apparent. The two Fc-constructs have a size of about 120 kDa, which can possibly indicate a dimeric form of the Fc-tag connected to one hemagglutinin. As described previously, it was not possible to detect the 1918_T6his in the supernatants of transfected HEK-293T cells (figure 27.A + 27.B). There are also many unspecific bands detectable with the anti-H1 antibody, which make an evaluation of Western Blots difficult. Furthermore, there are some bands in the pCG1 negative control under non-reducing conditions (figure 27.A), which can either be explained by an overflow of the sample from the adjacent pocket or a strong unspecific binding of the antibody, because the gel under reducing conditions (figure 27.B) shows no band.

The detection of the soluble proteins in the cell lysates of transfected HEK-293T cells showed that under non-reducing conditions (figure 28.C) several unspecific bands are detectable with the anti-H1 antibody with a size of 60 to 70 kDa. Under non-reducing conditions there is not much protein detectable in the cell lysates of 1918_sol and 1918_Fc. In the cell lysates of the other soluble proteins high molecular bands are detectable under non-reducing conditions (figure 28.C). The 1918_T6his protein, which could not be detected in the supernatants of transfected HEK-293T cells is visible at a size of more than 300 kDa and might therefore be present in a multimeric form (figure 28.C). Only the 2009_sol protein seems to display a trimeric form of about 240 kDa, whereas the 2009_Fc construct is additionally present in the form of a dimer (180 to 190 kDa). The negative control pCG1 does not show any additional band to the two nonspecific bands which can be explained by the unspecific binding of the anti-H1 antibody (figure 28.C).

Analysis of the cell lysates under reducing conditions (figure 28.D) showed again the presence of unspecific bands, which were detected in the Western Blot under non-reducing conditions. But additionally, bands with a molecular weight of about 75 kDa were detected in the different constructs without tag, with trimerization domain or with trimerization domain and his-tag (figure 28.D). Under reducing conditions, the

1918_Fc and the 2009_Fc construct exhibit a band with a size of approximately 110 to 120 kDa illustrating a monomeric form of the two different Fc-constructs (figure 28.D).

For further studies, the soluble 1918_Fc and the 2009_Fc proteins were used, because due to the Fc-tag purification via FPLC was accomplished conveniently by choosing Hi Trap™ Protein A HP columns.