SBT5.2s are the major active extracellular subtilases processing IgG antibody 2F5 in the Nicotiana benthamiana apoplast

Plants offer a powerful platform for recombinant protein production but degradation of recombinant proteins by endogenous proteases is causing severe yield losses. Here, we introduce triple knockout lines for SBT5.2, the major active subtilases in the apoplast of agroinfiltrated Nicotiana benthamiana. HIV-neutralising IgG antibody 2F5 is no longer cleaved in the apoplast of sbt5.2 mutants and these mutants accumulate 3-fold more 2F5 upon transient expression but grow normally. Remarkably, however, 2F5 does not accumulate in the apoplast and is not exposed to SBT5.2 when transiently expressed, uncovering an important controversy regarding the subcellular localisation of IgGs in agroinfiltrated plants.