Abstract
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 α1 -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.
Keywords:
Nicotiana benthamiana; biopharmaceutical; molecular farming; monoclonal antibody; recombinant protein expression; serine protease.
© 2020 Federation of European Biochemical Societies.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Agrobacterium tumefaciens / genetics
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Agrobacterium tumefaciens / metabolism
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Amino Acid Chloromethyl Ketones / pharmacology
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Antibodies, Monoclonal / biosynthesis
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Antibodies, Monoclonal / chemistry*
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Antibodies, Monoclonal / genetics
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Gene Expression
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HIV Antibodies / biosynthesis
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HIV Antibodies / chemistry*
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HIV Antibodies / genetics
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Isoenzymes / antagonists & inhibitors
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Isoenzymes / genetics
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Isoenzymes / metabolism
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Nicotiana / drug effects
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Nicotiana / enzymology
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Nicotiana / genetics*
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Phenylmethylsulfonyl Fluoride / pharmacology
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Plant Proteins / antagonists & inhibitors*
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Plant Proteins / genetics
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Plant Proteins / metabolism
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Plants, Genetically Modified
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Protease Inhibitors / pharmacology
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Proteolysis
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Recombinant Proteins / biosynthesis
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Recombinant Proteins / chemistry
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Recombinant Proteins / genetics
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Subtilisins / antagonists & inhibitors*
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Subtilisins / genetics
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Subtilisins / metabolism
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alpha 1-Antitrypsin / biosynthesis
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alpha 1-Antitrypsin / chemistry*
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alpha 1-Antitrypsin / genetics
Substances
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Amino Acid Chloromethyl Ketones
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Antibodies, Monoclonal
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HIV Antibodies
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Isoenzymes
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N-acetyl-tyrosyl-valyl-alanyl-aspartyl chloromethyl ketone
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Plant Proteins
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Protease Inhibitors
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Recombinant Proteins
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alpha 1-Antitrypsin
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benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone
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Phenylmethylsulfonyl Fluoride
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Subtilisins