Expression in Escherichia coli, Refolding, and Purification of Plant Aspartic Proteases.
Aspartic proteases
Atypical aspartic proteases
E. coli
Heterologous expression
Inclusion bodies
Nucellin-like aspartic proteases
Pepsin-like
Plant
Refolding
Typical aspartic proteases
Journal
Methods in molecular biology (Clifton, N.J.)
ISSN: 1940-6029
Titre abrégé: Methods Mol Biol
Pays: United States
ID NLM: 9214969
Informations de publication
Date de publication:
2022
2022
Historique:
entrez:
18
5
2022
pubmed:
19
5
2022
medline:
21
5
2022
Statut:
ppublish
Résumé
Aspartic proteases (APs) are widely distributed in plants. The large majority of genes encoding putative APs exhibit distinct features when compared with the so-called typical APs, and have been grouped as atypical and nucellin-like APs. Remarkably, a diverse pattern of enzymatic properties, subcellular localizations, and biological functions are emerging for these proteases, illustrating the functional complexity among plant pepsin-like proteases. However, many key questions regarding the structure-function relationships of plant APs remain unanswered. Therefore, the expression of these enzymes in heterologous systems is a valuable strategy to unfold the unique features/biochemical properties among members of this family of proteases. Here, we describe our protocol for the production and purification of recombinant plant APs, using a procedure where the protein is refolded from inclusion bodies by dialysis. This method allows the production of untagged versions of the target protease, which has revealed to be critical to disclose differences in processing/activation requirements between plant APs. The protocol includes protein expression, washing and solubilization of inclusion bodies, refolding by dialysis, and a protein purification method. Specific considerations on critical aspects of the refolding process and further suggestions for evaluation of the final recombinant product are also provided.
Identifiants
pubmed: 35583770
doi: 10.1007/978-1-0716-2079-3_3
doi:
Substances chimiques
Recombinant Proteins
0
Aspartic Acid Proteases
EC 3.4.-
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
21-33Informations de copyright
© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.
Références
Simoes I, Faro C (2004) Structure and function of plant aspartic proteinases. Eur J Biochem 271(11):2067–2075. https://doi.org/10.1111/j.1432-1033.2004.04136.x
doi: 10.1111/j.1432-1033.2004.04136.x
pubmed: 15153096
Soares A, Ribeiro Carlton SM, Simoes I (2019) Atypical and nucellin-like aspartic proteases: emerging players in plant developmental processes and stress responses. J Exp Bot 70(7):2059–2076. https://doi.org/10.1093/jxb/erz034
doi: 10.1093/jxb/erz034
pubmed: 30715463
Chen J, Ouyang Y, Wang L, Xie W, Zhang Q (2009) Aspartic proteases gene family in rice: gene structure and expression, predicted protein features and phylogenetic relation. Gene 442(1–2):108–118. https://doi.org/10.1016/j.gene.2009.04.021
doi: 10.1016/j.gene.2009.04.021
pubmed: 19409457
Faro C, Gal S (2005) Aspartic proteinase content of the Arabidopsis genome. Curr Protein Pept Sci 6(6):493–500
doi: 10.2174/138920305774933268
Guo R, Xu X, Carole B, Li X, Gao M, Zheng Y, Wang X (2013) Genome-wide identification, evolutionary and expression analysis of the aspartic protease gene superfamily in grape. BMC Genomics 14:554. https://doi.org/10.1186/1471-2164-14-554
doi: 10.1186/1471-2164-14-554
pubmed: 23945092
pmcid: 3751884
Castanheira P, Samyn B, Sergeant K, Clemente JC, Dunn BM, Pires E, Van Beeumen J, Faro C (2005) Activation, proteolytic processing, and peptide specificity of recombinant cardosin A. J Biol Chem 280(13):13047–13054. https://doi.org/10.1074/jbc.M412076200
doi: 10.1074/jbc.M412076200
pubmed: 15677463
Prasad BD, Creissen G, Lamb C, Chattoo BB (2010) Heterologous expression and characterization of recombinant OsCDR1, a rice aspartic proteinase involved in disease resistance. Protein Expr Purif 72(2):169–174. https://doi.org/10.1016/j.pep.2010.03.018
doi: 10.1016/j.pep.2010.03.018
pubmed: 20347986
Simoes I, Faro R, Bur D, Faro C (2007) Characterization of recombinant CDR1, an Arabidopsis aspartic proteinase involved in disease resistance. J Biol Chem 282(43):31358–31365. https://doi.org/10.1074/jbc.M702477200
doi: 10.1074/jbc.M702477200
pubmed: 17650510
Almeida CM, Pereira C, da Costa DS, Pereira S, Pissarra J, Simoes I, Faro C (2012) Chlapsin, a chloroplastidial aspartic proteinase from the green algae Chlamydomonas reinhardtii. Planta 236(1):283–296. https://doi.org/10.1007/s00425-012-1605-2
doi: 10.1007/s00425-012-1605-2
pubmed: 22349731
Bi X, Khush GS, Bennett J (2005) The rice nucellin gene ortholog OsAsp1 encodes an active aspartic protease without a plant-specific insert and is strongly expressed in early embryo. Plant Cell Physiol 46(1):87–98. https://doi.org/10.1093/pcp/pci002
doi: 10.1093/pcp/pci002
pubmed: 15659452
Gao H, Zhang Y, Wang W, Zhao K, Liu C, Bai L, Li R, Guo Y (2017) Two membrane-anchored aspartic proteases contribute to pollen and ovule development. Plant Physiol 173(1):219–239. https://doi.org/10.1104/pp.16.01719
doi: 10.1104/pp.16.01719
pubmed: 27872247
Ge X, Dietrich C, Matsuno M, Li G, Berg H, Xia Y (2005) An Arabidopsis aspartic protease functions as an anti-cell-death component in reproduction and embryogenesis. EMBO Rep 6(3):282–288. https://doi.org/10.1038/sj.embor.7400357
doi: 10.1038/sj.embor.7400357
pubmed: 15723040
pmcid: 1299267
Kadek A, Tretyachenko V, Mrazek H, Ivanova L, Halada P, Rey M, Schriemer DC, Man P (2014) Expression and characterization of plant aspartic protease nepenthesin-1 from Nepenthes gracilis. Protein Expr Purif 95:121–128. https://doi.org/10.1016/j.pep.2013.12.005
doi: 10.1016/j.pep.2013.12.005
pubmed: 24365662
Paparelli E, Gonzali S, Parlanti S, Novi G, Giorgi FM, Licausi F, Kosmacz M, Feil R, Lunn JE, Brust H, van Dongen JT, Steup M, Perata P (2012) Misexpression of a chloroplast aspartyl protease leads to severe growth defects and alters carbohydrate metabolism in Arabidopsis. Plant Physiol 160(3):1237–1250. https://doi.org/10.1104/pp.112.204016
doi: 10.1104/pp.112.204016
pubmed: 22987884
pmcid: 3490589
Soares A, Niedermaier S, Faro R, Loos A, Manadas B, Faro C, Huesgen PF, Cheung AY, Simoes I (2019) An atypical aspartic protease modulates lateral root development in Arabidopsis thaliana. J Exp Bot. https://doi.org/10.1093/jxb/erz059
Yao X, Xiong W, Ye T, Wu Y (2012) Overexpression of the aspartic protease ASPG1 gene confers drought avoidance in Arabidopsis. J Exp Bot 63(7):2579–2593. https://doi.org/10.1093/jxb/err433
doi: 10.1093/jxb/err433
pubmed: 22268147
pmcid: 3346222
Lin X, Koelsch G, Wu S, Downs D, Dashti A, Tang J (2000) Human aspartic protease memapsin 2 cleaves the beta-secretase site of beta-amyloid precursor protein. Proc Natl Acad Sci U S A 97(4):1456–1460. https://doi.org/10.1073/pnas.97.4.1456
doi: 10.1073/pnas.97.4.1456
pubmed: 10677483
pmcid: 26455
Burgess RR (2009) Refolding solubilized inclusion body proteins. Methods Enzymol 463:259–282. https://doi.org/10.1016/S0076-6879(09)63017-2
doi: 10.1016/S0076-6879(09)63017-2
pubmed: 19892177