Rhizosecretion improves the production of Cyanovirin-N in Nicotiana tabacum through simplified downstream processing

Abstract

Rhizosecretion has many advantages for the production of recombinant pharmaceuticals, notably facile downstream processing from hydroponic medium. The aim of this study was to increase yields of the HIV microbicide candidate, Cyanovirin-N (CV-N), obtained using this production platform and to develop a simplified methodology for its downstream processing from hydroponic medium. Placing hydroponic cultures on an orbital shaker more than doubled the concentration of CV-N in the hydroponic medium compared to plants which remained stationary, reaching a maximum of approximately 20μg/ml in one week, which is more than 3 times higher than previously reported yields. The protein composition of the hydroponic medium, the rhizosecretome, was characterised in plants cultured with or without the plant growth regulator alpha-napthaleneacetic acid by LC-ESI-MS/MS, and CV-N was the most abundant protein. The issue of large volumes in the rhizosecretion system was addressed by using ion exchange chromatography to concentrate CV-N and partially remove impurities. The semi-purified CV-N was demonstrated to bind to HIV gp120 in an ELISA and to neutralise HIVBa-L with an IC50 of 6nM in a cell-based assay. Rhizosecretion is therefore a practicable and inexpensive method for the production of functional CV-N.

Keywords: Cyanovirin-N; HIV microbicide; Hydroponic medium; Molecular pharming; Rhizosecretion.

Figure 1

Effect of continuous agitation of cultures on CV-N yields in hydroponic medium. Plants were grown for 6 weeks and then given fresh medium with NAA. Plants in the shaking group were then placed on an orbital shaker at 150 rpm and the control group remained stationary. Medium with NAA was replaced at weeks 8, 9 and 10. (A) Yields at weeks 7-11. Values are means ± SEM for a minimum of 8 plants. (B) Coomassie-stained SDS-PAGE gel of week 8 pooled samples from the stationary control and shaking groups. CT: stationary control, SH: shaking. Molecular weight is indicated on the left in kDa. (C) Root phenotypes of representative plants in the stationary control and shaking groups at week 10.

Figure 2

Relative abundance of protein classes in the rhizosecretome of plants cultured in MS medium with or without NAA. Hydroponic medium was analyzed by LC-ESI-MS/MS followed by a Mascot search with plant taxonomy parameter and a calculation of Exponentially Modified Protein Abundance Index.

Figure 3

CV-N semi-purification by IEX (A) Silver stained SDS-PAGE gel with samples from the first IEX run for purification of CV-N. (B) Western blot to detect CV-N in IEX Elution 1. (C) Silver-stained SDS-PAGE gel with samples from the second IEX run for purification of CV-N. Marker - Molecular weight protein markers, CV-N (100 ng) - purified E. coli CV-N positive control (100 ng), loaded sample - dialyzed hydroponic medium prior to column loading, flow-through – IEX column flow through.

Figure 4

Inhibition of HIV infection by rhizosecreted CV-N following IEX. Luciferase assays were performed by incubating the reporter cell line TZM-bl with HIV_BaL and rhizosecreted IEX-purified CV-N or E. coli derived CV-N (initial concentration of 1.75μg/ml and 10 μg/ml, respectively) or PBS. The viral infectivity is plotted against the dilution of the sample, and results are the mean and SEM of three experiments. The table indicates the IC₅₀ of rhizosecreted IEX-purified and E.coli-derived CV-N. Calculations were performed using GraphPad Prism software.