Chloroplast expression of His-tagged GUS-fusions: a general strategy to overproduce and purify foreign proteins using transplastomic plants as bioreactors

Abstract

High level expression and efficient recovery of recombinant protein aretwo main critical factors that determine the use of transgenic plants asnaturalbioreactors to produce foreign proteins for industrial applications. Wedemonstrate here the potential of a new strategy involving chloroplasttransformation, GUS-fusions and affinity-tag based chromatography tooverexpressand purify a human therapeutic protein, interferon gamma (IFN-g) in tobaccoplants. Our results show that IFN-g accumulation reaches up to 6% of totalsolubleprotein when expressed as a GUS-fusion protein in tobacco chloroplasts.Additionof His-tag simplified the downstream process and the recombinant protein yieldswere considerably high (∼360 μg/g fresh leaf tissue).Further we demonstrate the use of GUS-fusions to identify recombinant proteincontaining fractions very rapidly (< 5 minutes) through simple GUS assay, animportant consideration for those proteins that are highly labile duringlengthyand harsh downstream processing conditions. The chloroplast-produced IFN-g isbiologically as active as the same protein obtained through E.coli expression without any involvement of refolding procedure. Ourresults demonstrate that the new strategy has tremendous potential for largescale production of proteins from heterologous source, independent of theirphysio-chemical and biological properties, using plants as ‘naturalbioreactors’.