title: Agroinfiltration contributes to VP1 recombinant protein degradation.
abstract: There is a growing interest in applying tobacco agroinfiltration for recombinant protein production in a plant based system. However, in such a system, the action of proteases might compromise recombinant protein production. Protease sensitivity of model recombinant foot-and-mouth disease (FMD) virus P1-polyprotein (P1) and VP1 (viral capsid protein 1) as well as E. coli glutathione reductase (GOR) were investigated. Recombinant VP1 was more severely degraded when treated with the serine protease trypsin than when treated with the cysteine protease papain. Cathepsin L- and B-like as well as legumain proteolytic activities were elevated in agroinfiltrated tobacco tissues and recombinant VP1 was degraded when incubated with such a protease-containing tobacco extract. In silico analysis revealed potential protease cleavage sites within the P1, VP1 and GOR sequences. The interaction modeling of the single VP1 protein with the proteases papain and trypsin showed greater proximity to proteolytic active sites compared to modeling with the entire P1-polyprotein fusion complex. Several plant transcripts with differential expression were detected 24 hr post-agroinfiltration when the RNA-seq technology was applied to identify changed protease transcripts using the recently available tobacco draft genome. Three candidate genes were identified coding for proteases which included the Responsive-to-Desiccation-21 (RD21) gene and genes for coding vacuolar processing enzymes 1a (NbVPE1a) and 1b (NbVPE1b). The data demonstrates that the tested recombinant proteins are sensitive to protease action and agroinfiltration induces the expression of potential proteases that can compromise recombinant protein production.
authors.affiliation: a Department of Plant and Soil Sciences , Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria , Hillcrest, Pretoria , South Africa.
authors.forename: Priyen
authors.initials: P
authors.lastname: Pillay
authors.affiliation: a Department of Plant and Soil Sciences , Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria , Hillcrest, Pretoria , South Africa.
authors.forename: Karl J
authors.initials: KJ
authors.lastname: Kunert
authors.affiliation: a Department of Plant and Soil Sciences , Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria , Hillcrest, Pretoria , South Africa.
authors.forename: Stefan
authors.initials: S
authors.lastname: van Wyk
authors.affiliation: a Department of Plant and Soil Sciences , Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria , Hillcrest, Pretoria , South Africa.
authors.forename: Matome Eugene
authors.initials: ME
authors.lastname: Makgopa
authors.affiliation: b Department of Biology , Case Western Reserve University , Cleveland , OH , USA.
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Cullis
authors.affiliation: a Department of Plant and Soil Sciences , Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria , Hillcrest, Pretoria , South Africa.
authors.forename: Barend J
authors.initials: BJ
authors.lastname: Vorster
mesh_terms: D001673:Biodegradation, Environmental; D036022:Capsid Proteins / Q000096:biosynthesis / Q000235:genetics; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism; D057056:Cysteine Proteases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli; D029544:Foot-and-Mouth Disease Virus; D020869:Gene Expression Profiling; D018506:Gene Expression Regulation, Plant; D005818:Genetic Engineering; D005980:Glutathione Reductase / Q000096:biosynthesis / Q000235:genetics; D058465:Nitrification; D018515:Plant Leaves / Q000737:chemistry; D030821:Plants, Genetically Modified / Q000737:chemistry / Q000235:genetics; D018749:RNA, Plant / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D017423:Sequence Analysis, RNA; D057057:Serine Proteases / Q000235:genetics / Q000378:metabolism*; D014026:Nicotiana / Q000737:chemistry / Q000235:genetics*
chemical_list: D036022:Capsid Proteins; D018749:RNA, Plant; D011994:Recombinant Proteins; D005980:Glutathione Reductase; D057056:Cysteine Proteases; D057057:Serine Proteases; D003546:Cysteine Endopeptidases; C089278:vacuolar processing enzyme
keywords: VP1 protein; agroinfiltration; cysteine proteases; foot-and-mouth disease; proteases; recombinant protein production; tobacco