Recombinant expression, purification, and functional analysis of two novel cystatins from sugarcane (Saccharum officinarum)

Recombinant expression, purification, and functional analysis of two novel cystatins from sugarcane (Saccharum officinarum)

Autor Gianotti, A. Google Scholar
Rios, W. M. Google Scholar
Soares-Costa, A. Google Scholar
Nogaroto, V Google Scholar
Carmona, A. K. Google Scholar
Oliva, MLV Google Scholar
Andrade, S. S. Google Scholar
Henrique-Silva, F. Google Scholar
Instituição Universidade Federal de São Carlos (UFSCar)
Universidade Federal de São Paulo (UNIFESP)
Resumo 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 (similar to 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 mu M 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. (c) 2005 Elsevier Inc. All rights reserved.
Palavra-chave cysteine proteinase inhibitor
cystatin
phytocystatin
canecystatin
sugarcane
Saccharum officinarum
Idioma Inglês
Data de publicação 2006-06-01
Publicado em Protein Expression and Purification. San Diego: Academic Press Inc Elsevier Science, v. 47, n. 2, p. 483-489, 2006.
ISSN 1046-5928 (Sherpa/Romeo, fator de impacto)
Publicador Elsevier B.V.
Extensão 483-489
Fonte http://dx.doi.org/10.1016/j.pep.2005.10.026
Direito de acesso Acesso restrito
Tipo Artigo
Web of Science WOS:000238277000020
Endereço permanente http://repositorio.unifesp.br/handle/11600/28956

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