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Rhizosecretion improves the production of Cyanovirin‐N in Nicotiana tabacum through simplified downstream processing
Author(s) -
Madeira Luisa M.,
Szeto Tim H.,
Ma Julian KC.,
Drake Pascal M.W.
Publication year - 2016
Publication title -
biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.144
H-Index - 84
eISSN - 1860-7314
pISSN - 1860-6768
DOI - 10.1002/biot.201500371
Subject(s) - downstream processing , recombinant dna , chemistry , chromatography , nicotiana tabacum , downstream (manufacturing) , biochemistry , gene , operations management , economics
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 HIV Ba‐L with an IC 50 of 6nM in a cell‐based assay. Rhizosecretion is therefore a practicable and inexpensive method for the production of functional CV‐N.