Identification of two subtilisin-like serine proteases engaged in the degradation of recombinant proteins in Nicotiana benthamiana

The tobacco variant Nicotiana benthamiana has recently emerged as a versatile host for the manufacturing of protein therapeutics, but the fidelity of many recombinant proteins generated in this system is compromised by inadvertent proteolysis. Previous studies have revealed that the anti-HIV-1 antibodies 2F5 and PG9 as well as the protease inhibitor alpha(1)-antitrypsin (A1AT) are particularly susceptible to N. benthamiana proteases. Here, we identify two subtilisin-like serine proteases (NbSBT1 and NbSBT2) whose combined action is sufficient to account for all major cleavage events observed upon expression of 2F5, PG9 and A1AT in N. benthamiana. We propose that downregulation of NbSBT1 and NbSBT2 activities could constitute a powerful means to optimize the performance of this promising platform for the production of biopharmaceuticals. Databases NbSBT sequence data are available in the DDBJ/EMBL/GenBank databases under the accession numbers MN534996 to MN535005.

Automated summary

Nicotiana benthamiana is a versatile host for manufacturing protein therapeutics, but the fidelity of recombinant proteins is compromised by proteolysis. Two subtilisin-like serine proteases (NbSBT1 and NbSBT2) have been identified as the main causes of proteolysis, suggesting that downregulating their activities could optimize the production of biopharmaceuticals on this platform. Sequence data of NbSBT are available in the DDBJ/EMBL/GenBank databases.