Enhanced-Transient Expression of Hepatitis C Virus Core Protein in Nicotiana tabacum, a Protein With Potential Clinical Applications

Mohammadzadeh, Sara; Khabiri, Alireza; Roohvand, Farzin; Memarnejadian, Arash; Salmanian, Ali Hatef; Ajdary, Soheila; Ehsani, Parastoo

Coomassie-stained SDS-PAGE showed that nonspecifically-bound plant proteins were eluted from Ni-NTA affinity chromatography besides a band with an electrophoretic mobility of 15 kDa (Figure 4 B) . The recent band was visible on a Coomassie-stained SDS-PAGE gel (Figure 4 B) and was also detected using anticore polyclonal antibodies in a western blot assay. There was no degradation of pHCVcp and the protein was not glycosylated because the size of the produced protein was as predicted by the protein sequence (https://www.genscript. com/ssl-bin/site2/peptide_calculation.cgi). As shown in Figure 4 C, the results of western blot analysis indicated that pHCVcp from PVX-core and pBI-core expressed a protein of approximately 15 kDa, while the negative control lacked the core protein and eHCVcp showed a higher molecular weight (~21 kDa, which is due to addition of vector-derived amino acids in pIVEX2.4a plasmid) and a few other lower-molecular weight protein bands which were presumably the results of ribosomal release and uncompleted translation, as previously reported (15) . It should be noted that dot blotting in this study was performed based on prior studies that used this procedure in complementation of western blotting (7, 8) . Indeed, In western blotting, the protein will be completely denatured (by boiling in SDS-PAGE loading buffer which contains 2-ME and SDS) and therefore some information that might be related to the native/soluble structure of the protein might be lost (e.g. interaction of protein with a conformational antibody), while dot blot assay was used as a preliminary and fast test to show if the protein was expressed.