Abstract:
The hypersensitive response is elicited by Agrobacterium infiltration of
Nicotiana benthamiana, including the induction and accumulation of
pathogenesis-related proteins, such as proteases. This includes the induction
of the expression of several cysteine proteases from the C1 (papain-like
cysteine protease) and C13 (legumain-like cysteine protease) families. This
study demonstrates the role of cysteine proteases: NbVPE-1a, NbVPE-1b,
and NbCysP6 in the proteolytic degradation of Nicotiana benthamiana
(glycosylation mutant 1XTFT)-produced anti-human immunodeficiency virus
broadly neutralizing antibody, CAP256-VRC26.25. Three putative cysteine
protease cleavage sites were identified in the fragment crystallizable region.
We further demonstrate the transient coexpression of CAP256-VRC26.25
with CRISPR/Cas9-mediated genome editing vectors targeting the
NbVPE-1a, NbVPE-1b, and NbCysP6 genes which resulted in a decrease
in CAP256-VRC26.25 degradation. No dierences in structural features were
observed between the human embryonic kidney 293 (HEK293)-produced
and 1XTFT broadly neutralizing antibodies produced with and without the
coexpression of genome-editing vectors. Furthermore, despite the presence
of proteolytically degraded fragments of plant-produced CAP256-VRC26.25
without the coexpression of genome editing vectors, no influence on
the in vitro functional activity was detected. Collectively, we demonstrate
an innovative in planta strategy for improving the quality of the CAP256
antibodies through the transient expression of the CRISPR/Cas9 vectors.
