Recombinant expression, purification, and functional analysis of two novel cystatins from sugarcane (Saccharum officinarum) |
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Authors: | Gianotti Andréia Rios Wendy M Soares-Costa Andrea Nogaroto Viviane Carmona Adriana K Oliva Maria L V Andrade Sheila S Henrique-Silva Flávio |
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Affiliation: | Laboratório de Biologia Molecular, DGE, Universidade Federal de S?o Carlos, Rodovia Washington Luis km 235, SP, Brazil. |
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Abstract: | Phytocystatins are cysteine proteinase inhibitors from plants implicated in the endogenous regulation of protein turnover, programmed cell death, and in defense mechanisms against pathogens. To date, only few cystatin genes have been characterized in most plant species. We have previously characterized the protein Canecystatin, the first cystatin described in sugarcane. In an attempt to study novel Canecystatins, we identified two ORFs encoding cystatins (referred as CaneCPI-2 and CaneCPI-3) using the data from the Sugarcane EST genome project. These ORFs were then subcloned and expressed in Escherichia coli using pET28 expression vector. High amounts (approximately 20 mg/L) of pure recombinant proteins were obtained by affinity chromatography in a single step of purification. Polyclonal antibodies against the recombinant Canecystatins were raised, allowing the immunodetection of the endogenous proteins in the plant tissues. Moreover, the proteins were able to inhibit papain in a fluorometric assay with K(i) values of 0.2 and 0.25 microM for CaneCPI-2 and CaneCPI-3, respectively. These findings contribute to a better understanding of the activity of sugarcane cystatins and encourage future activity and structural studies of these proteins. |
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Keywords: | Cysteine proteinase inhibitor Cystatin Phytocystatin Canecystatin Sugarcane Saccharum officinarum |
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