Bulk production of the antiviral lectin griffithsin

Fuqua, Joshua L.; Hamorsky, Krystal; Khalsa, Guruatma; Matoba, Nobuyuki; Palmer, Kenneth E.

After successful production and qualification of the plasmid bank, it is used to transcribe infectious RNA for inoculation of a number of plants, which is determined based on the size of the bank you are producing (the final virion volume will be approximately 10% of the harvested biomass). The plants are harvested relatively early in the normal production process, approximately day 5 after infection. The virion amplified in the plant tissue is extracted, isolated and concentrated using a modified polyethylene glycol precipitation procedure and stored at À20°C (Asurmendi et al., 2007) . An approximate concentration of the virion can be determined through UV absorption at 260 nm with an extinction coefficient of 3.0 (Kathiria et al., 2010) . The isolated virion is qualified for use through functional assessment by application of 50 ng of virus per plant with a mild abrasive (Kathiria et al., 2010) . Function is assessed by display of symptoms characteristic of rTMV vector infection and GRFT expression level with the same criteria required as with 'naked' RNA, 90% symptomatic plants and >200 mg/kg of GRFT. For comparison to prior work with TMV, electron microscopy images were used to visualize TMV particles and data were compared to historical information regarding TMV virion size ( Figure 1c ). Upon qualification of the virion bank, a stability program will be implemented ( Table 1 ). The stability program mirrors the qualification program, but is carried out over a 2-year period as the virion bank is being stored at À20°C.