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

The primary objective of the present study was to provide an efficient transient tobacco system for expression of HCVcp in a regionally-adapted tobacco host (the Iranian Jafarabadi-tobacco plant cultivar). While other tobacco plant such as Australian Nicotiana species, N. benthamiana, is a well-known and hyper-susceptible host for plant-derived recombinant protein expression (47) , to our knowledge there was no prior report available on expression in Jafarabadi tobacco cultivar (which is one of the currently available tobacco cultivar in Iran). To address this concern, the first strategy that should be considered would be the optimization of the gene sequence for efficient codon usage by the plant (27) . In agreement, it is recently shown that the plant codonoptimized BPV-1 LI gene product increased significantly in comparison to the unmodified counterpart (28) . As shown in Figure 2 A, our codon optimization approach increased the CAI value from 0.65 to 0.85, which was in favor of codon utilization for N. tabacum (35) . Although increasing the CAI value is usually inevitably accompanied by increase of A + T percentage (which enhances mRNA instability and decreases the efficiency of translation) (48, 49) , our codon optimization approach that was based on the codon usage of nuclear-encoded genes of tobacco could keep the A + T percentage at 49%, while reducing the GC content from 62.62 to 51.05 (Figure 2 B) , almost the perfect range desired for GC content (50%). To ameliorate the expression level, insertion of the Kozak sequence (GCCACCATGGC) (36) at the translation start site (ATG) was also considered ( Figure 1 ). However, since the two base pairs immediately following the ATG start codon were "C" and "A" and were not compatible with the optimized Kozak sequence (underlined nucleotides), consistent with the approach undertaken by Amani et al. (50) , we had to insert the GCT sequence that codes for alanine (a nonpolar amino acid). Moreover, to improve the stability of pHCVcp, KDEL-encoded bases were also considered at the 3' site of the Tr-HCVcp sequence ( Figure 1 ). Employing this strategy for expression of human epidermal growth factor in tobacco resulted in a 10 4 -fold higher yield of protein expression (51) . Finally, in our codon optimization strategy, the possible deleterious splicing motif in plants "GGTAAG", resulting in RNA degradation and gene silencing of the gene (52) , was removed from the native HCVcp-coding sequence (nucleotides 358-363).