Design, production and characterization of light and heavy chain of human fix produced in N. benthamiana

Hemophilia B is an inherited disease caused by a mutation on X chromosome that result the deficiency of factor IX protein in blood. The Factor IX protein undergoes a two-step proteolytic cleavage to be activated. Two chain inactive Factor IXa are formed with interruption on Arg145. Following this process, hydrolysis occurs in Arg180 region and the disulphide bonds between the N-terminal containing the light chain (LC) and the C-terminus containing the heavy chain (HC) are formed, and the active protease Factor IXab was present. Activated Factor IX contains of four structural domains: the carboxyglutamic acid (Gla) domain as the N-terminus, two epidermal growth factor-like domains and finally the proteolytic domain. Currently, patients with hemophilia B are treated either by obtaining FIX in plasma from human blood or by recombinant FIX produced in CHO (Chinese hamster ovary) cells. Studies have shown recombinant FIX has also been successfully produced in mammalian cell cultures. However, this system extremely expensive and difficult to scale up Plant expression system is promising expression system and has a number of advantages compared to other expression systems. The goal of this study was to attempt the production of factor IX light and heavy chain domains by transient gene expression method in plant for possible of assembly in vivo. In this study, HC and LC genes were engineered and codon optimized using N. benthamiana codons. Codon optimized HC and LC genes were de novo synthesized and then cloned into plant expression vector. Cloned plasmids were transferred into Agrobacterium tumefaciens starin AGL1 by electroporation. Nicotiana benthamiana plants were infiltrated by agro harboring HC and LC genes. After confirmation the expression and optimization, both genes were introduced into plant cells by agroinfiltration for possible assembly of HC and LC. Plant infiltrated with LC and HC genes were harvested, extracted, purified and performed FIX activity. The activity was not higher than 10% relative to human Furin.