Recombinant HA protein expression and purification
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. Soluble HA proteins were constructed using the HA cDNA sequences of H5N1 A/Thailand/1(KAN-1)/2004. The PQRERRRKKRG multibasic protease cleavage site between HA1 and HA2 was mutated to PQRETRG to retain the uncleaved protein. The C-terminal sequence was fused with a GCN4pII leucine zipper sequence and ended with a His-tag to facilitate purification. Recombinant H5HA proteins were purified from the culture supernatants of Sf9, Mimic, and CHO cells and analyzed by SDS-PAGE with (A) Coomassie Blue staining and (B) western blotting with anti-H5 antibodies. Proteins were also analyzed following treatment with (C) Endo H and (D) PNGase F.