Agroinfiltration contributes to VP1 recombinant protein degradation

Show simple item record Pillay, Priyen Kunert, Karl J. Van Wyk, Stefan George Makgopa, M.E. (Matome Eugene) Cullis, C.A. (Christoper Ashley) Vorster, Barend Juan 2016-09-12T08:21:44Z 2016-08
dc.description.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 modelling of the single VP1 protein with the proteases papain and trypsin showed greater proximity to proteolytic active sites compared to modelling 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. en_ZA
dc.description.department Plant Science en_ZA
dc.description.embargo 2017-08-31
dc.description.librarian hb2016 en_ZA
dc.description.sponsorship The National Research Foundation (NRF) and the Genomics Research Institute (GRI), South Africa as well as NRF incentive funding to Karl Kunert and a NRF bursary to Priyen Pillay. en_ZA
dc.description.uri en_ZA
dc.identifier.citation Pillay, P, Kunert, KJ,Van Wyk, SG, Makgopa, ME, Cullis, CA & Vorster, BJ 2016, 'Agroinfiltration contributes to VP1 recombinant protein degradation', Bioengineered, vol. 7, no. 6, pp. 459-477. en_ZA
dc.identifier.issn 1949-1018 (print)
dc.identifier.issn 1949-1026 (online)
dc.identifier.other 10.1080/21655979.2016.1208868
dc.language.iso en en_ZA
dc.publisher Routledge en_ZA
dc.rights © Taylor and Francis. This is an electronic version of an article published in Bioengineered, vol. 7, no. 6, pp. 459-477, 2016. doi : 10.1080/21655979.2016.1208868. Bioengineered is available online at : en_ZA
dc.subject Agroinfiltration en_ZA
dc.subject Recombinant protein production en_ZA
dc.subject Proteases en_ZA
dc.subject Cysteine proteases en_ZA
dc.subject Tobacco en_ZA
dc.subject Foot-and-mouth disease (FMD) en_ZA
dc.subject Virus P1-polyprotein (P1) en_ZA
dc.subject Glutathione reductase (GOR) en_ZA
dc.subject Viral capsid protein 1 (VP1) en_ZA
dc.title Agroinfiltration contributes to VP1 recombinant protein degradation en_ZA
dc.type Postprint Article en_ZA

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