An endoplasmic reticulum (ER)-directed fusion protein comprising a bacterial subtilisin domain and the human cytokine interleukin 6 is efficiently cleaved in planta

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 eXact super(TM) 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 benthamiana leaves using the MagnlCON 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 eXact super(TM) system reduced the yield still further. The inefficient production of tagged IL6, coupled with the extensive losses during purification, indicate that the Profinity eXact super(TM) system is not suitable for the extraction of IL6 from crude plant extracts.