Abstract
SummaryPlant‐based platforms are extensively used for the expression of recombinant proteins, including monoclonal antibodies. However, to harness the approach effectively and leverage it to its full potential, a better understanding of intracellular processes that affect protein properties is required. In this work, we examined vacuolar (vac) targeting and deposition of the monoclonal antibody (Ab) 14D9 in Nicotiana benthamiana leaves. Two distinct vacuolar targeting signals (KISIA and NIFRGF) were C‐terminal fused to the heavy chain of 14D9 (vac‐Abs) and compared with secreted and ER‐retained variants (sec‐Ab, ER‐Ab, respectively). Accumulation of ER‐ and vac‐Abs was 10‐ to 15‐fold higher than sec‐Ab. N‐glycan profiling revealed the predominant presence of plant typical complex fucosylated and xylosylated GnGnXF structures on sec‐Ab while vac‐Abs carried mainly oligomannosidic (Man 7‐9) next to GnGnXF forms. Paucimannosidic glycans (commonly assigned as typical vacuolar) were not detected. Confocal microscopy analysis using RFP fusions showed that sec‐Ab‐RFP localized in the apoplast while vac‐Abs‐RFP were exclusively detected in the central vacuole. The data suggest that vac‐Abs reached the vacuole by two different pathways: direct transport from the ER bypassing the Golgi (Ab molecules containing Man structures) and trafficking through the Golgi (for Ab molecules containing complex N‐glycans). Importantly, vac‐Abs were correctly assembled and functionally active. Collectively, we show that the central vacuole is an appropriate compartment for the efficient production of Abs with appropriate post‐translational modifications, but also point to a reconsideration of current concepts in plant glycan processing.
Highlights
The synthesis of pharmaceutical and industrial proteins in plants has became a reality with numerous products on the market and a variety of technologies available and facilities installed for largescale production (Sack et al, 2015)
The plant central vacuole is considered a hostile environment for foreign protein accumulation, it has been shown that some proteins, such as human alpha-mannosidase (De Marchis et al, 2013), human complement factor C5a, (Nausch et al, 2012), collagen (Stein et al, 2009) and human transglutaminase 2 (Marin Viegas et al, 2015), accumulate in this organelle
We studied the impact of vacuolar deposition of an IgG antibody in Nicotiana benthamiana leaves
Summary
The synthesis of pharmaceutical and industrial proteins in plants has became a reality with numerous products on the market and a variety of technologies available and facilities installed for largescale production (Sack et al, 2015). Stable and transient expression systems, have been successfully used to express mAbs in different plant species, organs and subcellular compartments (De Muynck et al, 2010). Recombinant protein accumulation levels rely on numerous factors among them subcellular localization (Egelkrout et al, 2012). Alternative strategies to optimize protein accumulation in other cellular organelles are considered. The plant central vacuole is considered a hostile environment for foreign protein accumulation, it has been shown that some proteins, such as human alpha-mannosidase (De Marchis et al, 2013), human complement factor C5a, (Nausch et al, 2012), collagen (Stein et al, 2009) and human transglutaminase 2 (Marin Viegas et al, 2015), accumulate in this organelle. Accumulation of full-length antibodies sorted to vacuoles in leaves has not been studied
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