Secretion of biologically active glycoforms of bovine follicle stimulatinghormone in plants

We chose the follicle stimulating hormone (FSH), a pituitary heterodimeric glycoprotein hormone, as a model to assess the ability of the plant cell to express a recombinant protein that requires extensive N-glycosylation for subunit folding and assembly, intracellular trafficking, signal transductio n and circulatory stability. A tobacco mosaic virus (TMV) based transient e xpression system was used to express a single-chain (sc) version of bovine FSH in the tobacco related species Nicotiana benthamiana. Preparations of p eriplasmic proteins from plants infected with recombinant viral RNA contain ed high levels of sc-bFSH, up to 3% of total soluble proteins. Consistently , in situ indirect immunofluorescence revealed that the plant cell secreted the mammalian secretory protein to the extracellular compartment (EC). By mass spectrometric analysis of immunoaffinity purified sc-bFSH derived from EC fractions, we found two species of the plant paucimannosidic glycan typ e, truncated forms of complex-type N-glycans. Stimulation of cAMP productio n in a CHO cell line expressing the porcine FSH receptor acknowledged the n ative-like structure of sc-bFSH and a sufficient extent of N-glycosylation required for signal transduction. Furthermore, in superovulatory treatments of mice, sc-bFSH displayed significant in vivo bioactivity, although much lower than that of pregnant mare serum gonadotropin. We conclude that plant s may have a broad utility as hosts for the recombinant expression of prote ins even where glycosylation is essential for function.