## 3 Results
### Functional and Structural Characterization of the Purified Haemagglutinin (HA) Proteins  
To precisely detect the function and structure of the recombinant artificial H5.c1 and native H5 TG proteins, which were transiently expressed in _N. benthamiana_, their purification is essential. These HA proteins were purified by IMAC. IMAC is a commonly employed method to purify recombinant proteins containing polyhistidine amino acid residues based on the strong specific interaction between these amino acids and immobilized metal ions (Co2+, Ni2+, Cu2+, and Zn2+) [32]. To obtain HA proteins from transiently transformed _N. benthamiana_ leaves, these proteins were expressed as fusion proteins containing a N-terminal 6 xHis-tag. The plant extract containing HA proteins was then incubated with Ni-NTA agarose. The HA proteins containing sequences of consecutive histidine residues were efficiently retained on IMAC column matrices. For washing and elution steps in the IMAC column matrices, 5 and 50 mM imidazole were tested for the artificial H5.c1 protein, while 30 and 125 mM imidazole were used for the native H5 TG, respectively [24]. Optimization of imidazole concentration for purification of H5.c1 by IMAC was shown in Figure S1. The IMAC-purified artificial H5.c1 protein was compared to the IMAC-purified native H5 TG protein for functional and structural characterization, as shown in Figure 4. The results showed that both of these HA proteins had a similar haemagglutination function, with only 0.23 ug of the purified HA proteins stopping the agglutination of rooster blood cells (as shown in Figure 4a). The trimeric HA exhibits epitopes that could induce neutralizing antibodies; therefore, it is important to retain the stable trimeric HA state [20]. In this study, the trimerization domain (GCN4-pII) was used to produce stable homotrimeric HA proteins. The multimeric states of IMAC-purified HA proteins were determined by cross-linking using BS3, a homobifunctional and water-soluble crosslink [33; 34]. In the previous study, the oligomeric status of H5 protein fused GCN4-pII was demonstrated by the cross-linking reaction with BS3, and the gel mobility of (H5 pII)3 was approximately consistent with the expected size of a trimer [21]. Similarly, analysis by denaturing SDS-PAGE and Western blot indicated that the recombinant artificial H5.c1 and native H5 TG proteins were observed as a mixture of multimeric protein without BS3 (as shown in Figure 4b, lanes 1, 3) and only trimeric  
Figure 4: Functional characterization of the immobilized metal ion affinity chromatography (IMAC)-purified haemagglutinin (HA) proteins (**a**); structural characterization of the IMAC-purified HA proteins by a cross-linking reaction using bis[sulfosuccinidyl] suberate (BS3) (**b**). proteins with BS3 (as shown in Figure 4, lanes 2, 4). These results were also similar to the study of [PERSON] and colleagues. This result indicated that the modification of the HA proteins at the C-terminus with the GCN4-pII motif stabilized the trimeric structure of the secreted recombinant HA protein [19]. The results were obtained when characterizing the structure of IMAC-purified artificial H5.c1 and native H5 TG proteins by SEC, as shown in Figure 5. The HA proteins presented from A11 to B7 fractions, in which most of the HA proteins eluted in the B1 to B6 fractions, which were fractions containing standard proteins with high molecular weights (160-500 kDa). This result indicated that most of the purified H5 proteins were oligomeric. The purified oligomeric H5 proteins from the B1 to B6 fractions were collected and then used for ELISA and Western blotting to detect HA-specific IgG mouse antibody production. The purity of collected fractions were confirmed by SDS-PAGE and Coomassie stain (Figure S2). Figure 5: Structural characterization of the purified haemagglutinin (HA) proteins by size exclusion chromatography (SEC) and the A9–B9 SEC fractions analyzed by SDS–PAGE 10% under reducing conditions and Western blot using an anti–c–myc antibody.