Production of a His-Tagged Canecystatin in Transgenic Sugarcane and Subsequent Purification

Autor(es)

Ribeiro, Carolina W. ^[1] ; Soares-Costa, Andrea ^[1] ; Falco, Maria Cristina ^[2] ; Chabregas, Sabrina M. ^[2] ; Ulian, Eugenio C. ^[2] ; Cotrin, Simone S. ^[3] ; Carmona, Adriana K. ^[3] ; Santana, Lucimeire A. ^[3] ; Oliva, Maria Luiza V. ^[3] ; Henrique-Silva, Flavio ^[1]

Afiliação do(s) autor(es)

^[1] Univ Fed Sao Carlos, Mol Biol Lab, Dept Genet & Evolut, BR-13565905 Sao Carlos, SP - Brazil

^[2] Ctr Tecnol Canavieira, BR-13400970 Piracicaba, SP - Brazil

^[3] Univ Fed Sao Paulo, Escola Paulista Med, BR-04044020 Sao Paulo - Brazil

• Tipo de documento: Artigo Científico
• Fonte: BIOTECHNOLOGY PROGRESS; v. 24, n. 5, p. 1060-1066, SEP-OCT 2008.
• Citações Web of Science: 8

Resumo

Transgenic plants have been used widely as expression systems of recombinant proteins in recent years. This process can be an efficient alternative for the large-scale production of proteins. In this work, we Present the establishment of transgenic sugarcane expressing a His-tagged canecystatin under the control of the maize ubiquitin promoter. A number of studies have demonstrated that cystatins, which are natural inhibitors of cysteine proteinases, can be used for protection against insect attacks. A transformed sugarcane plant that presented high levels of (HIS)CaneCPI-1 expression, was selected for the purification of this protein through affinity chromatography in a nickel column. This purified (HIS)CaneCPl-1 was immnunodetected using a polyclonal antibody, which was also able to detect the (HIS)CaneCPI-1 in a crude extract from transgenic plant leaves. Assays of inhibitory activity performed with the purified (HIS)CaneCPI-1 revealed its ability to inhibit the catalytic activity of midgut cysteine proteinase partially purified from the sugarcane weevil Sphenophorus levis and human cathepsin L in nanomolar order. These studies demonstrate that sugarcane is a viable expression system for recombinant protein production. (AU)

• Processo FAPESP: 98/14138-2 - Center for Structural Molecular Biotechnology
• Beneficiário: Glaucius Oliva
• Modalidade de apoio: Auxílio à Pesquisa - Centros de Pesquisa, Inovação e Difusão - CEPIDs
• Processo FAPESP: 05/59833-5 - Desenvolvimento de canas-de-acucar transgenicas superexpressando cistatinas visando o aumento da resistencia a insetos.
• Beneficiário: Andrea Soares da Costa Fuentes
• Modalidade de apoio: Bolsas no Brasil - Pós-Doutorado