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Protein Expr Purif. 2018 Jun;146:78-84. doi: 10.1016/j.pep.2018.01.004. Epub 2018 Jan 31.

Cloning, expression and purification of kinase domains of cacao PR-1 receptor-like kinases.

Author information

1
Instituto Agronômico, Campinas, SP, Brazil.
2
Structural Genomics Consortium, University of Campinas (SGC-UNICAMP), Campinas, SP, Brazil.
3
Structural Genomics Consortium, University of Campinas (SGC-UNICAMP), Campinas, SP, Brazil; Center for Molecular Biology and Genetic Engineering, University of Campinas (CBMEG-UNICAMP), Campinas, SP, Brazil.
4
Structural Genomics Consortium, University of Campinas (SGC-UNICAMP), Campinas, SP, Brazil; Center for Molecular Biology and Genetic Engineering, University of Campinas (CBMEG-UNICAMP), Campinas, SP, Brazil. Electronic address: rafael.counago@unicamp.br.
5
Instituto Agronômico, Campinas, SP, Brazil. Electronic address: jmcmondego@iac.sp.gov.br.

Abstract

The PR-1 proteins (pathogenesis-related protein 1) are involved in plant defense mechanisms against various pathogens. The genome of cacao (Theobroma cacao) encodes 14 PR-1 proteins, named TcPR-1a to TcPR-1n. Two of them, TcPR-1f and TcPR-1g, have a C-terminal expansion with high similarity to protein kinase domains, suggesting a receptor-like kinase (RLK) protein architecture. Moreover, TcPR-1g is highly expressed during cacao response to Witches' Broom Disease, caused by the fungus Moniliopthora perniciosa. Here we describe a structural genomics approach to clone, express and purify the kinase domains of TcPR-1f and TcPR-1g. Escherichia coli BL21(DE3)-R3 cells were used for protein expression and co-expression of Lambda Protein Phosphatase was critical for successfully obtaining soluble recombinant protein. We expect that the ability to express and purify the kinase domains of TcPR-1f and TcPR-1g will further our understanding of the role these proteins play during cacao defense response.

KEYWORDS:

Cacao; Ligation-independent cloning; Pathogenesis-related protein 1; Protein expression; Receptor-like protein kinase

PMID:
29360581
DOI:
10.1016/j.pep.2018.01.004
[Indexed for MEDLINE]

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