Abstract
A major limitation of plant bioreactors is the lack of suitable and cost-effective purification methods for the extraction of pharmaceutical-grade proteins. In contrast to that, there are numerous established purification systems for heterologous proteins, expressed in Escherichia coli, which are used for the commercial production of therapeutic proteins. Therefore, we wanted to adapt the BioRad Profinity eXactTM one-step protein purification system (originally designed for microbial expression platforms) to purify recombinant proteins in crude plant extracts. This system based on the prodomain of microbial subtilase as fusion partner and a column-bound subtilisin protease. The engineered protease captures and cleaves the fusion protein, retaining the tag and releasing the native protein into the eluate. The subtilase tag was fused to human interleukin 6 (IL6) and transiently expressed in Nicotiana benthamianaleaves using the MagnICON system. The fusion protein was expressed at lower levels than native IL6, suggesting it is expressed less efficiently and/or has a lower stability. However, free IL6 was also detected in the extract and was unaffected by the addition of protease inhibitors during extraction, suggesting that the fusion protein is cleaved in planta by endogenous proteases. Purification of the recombinant protein using the Profinity eXactTM system reduced the yield still further. The inefficient production of tagged IL6, coupled with the extensive losses during purification, indicate that the Profinity eXactTM system is not suitable for the extraction of IL6 from crude plant extracts. Key words: Tobacco, transient expression, endoplasmic reticulum, Profinity protein purification, partial cleavage.
Highlights
The production of pharmaceutical proteins in plants is attractive because of the anticipated lower costs, greater scalability and increased safety compared to traditional microbial and mammalian bioreactors (Davies, 2010).Abbreviations: interleukin 6 (IL6), Interleukin 6; Enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunosorbent assay.Many recombinant therapeutic proteins have been expressed in transgenic plants to demonstrate proof-ofconcept and there have been significant improvements in yields (Sharma and Sharma, 2009)
In order to test the Profinity eXactTM protein purification system we designed two constructs (Figure 1) which were introduced into the MagnICON vector pICH29912 for transient expression in tobacco
The threonine-serine linker is recommended by the manufacturers of the Profinity eXactTM protein purification system, these two amino acids remain on the purified protein after cleavage
Summary
The production of pharmaceutical proteins in plants is attractive because of the anticipated lower costs, greater scalability and increased safety compared to traditional microbial and mammalian bioreactors (Davies, 2010).Many recombinant therapeutic proteins have been expressed in transgenic plants to demonstrate proof-ofconcept and there have been significant improvements in yields (Sharma and Sharma, 2009). The production of pharmaceutical proteins in plants is attractive because of the anticipated lower costs, greater scalability and increased safety compared to traditional microbial and mammalian bioreactors (Davies, 2010). Downstream processing costs remain a major constraint for the commercial development of plant-derived pharmaceuticals (Wilken and Nikolov, 2012). Tobacco leaves contain more than 2500 compounds, including many alkaloids, phenolics and terpenoids (Nugroho and Verpoorte, 2002), which interfere with downstream processing (Wilken and Nikolov, 2012).
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