title: Production of a His-tagged canecystatin in transgenic sugarcane and subsequent purification.
abstract: 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)CaneCPI-1 was immunodetected 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.
authors.affiliation: Dept of Genetic and Evolution, Laboratory of Molecular Biology, Federal University of São Carlos, São Carlos, Brazil.
authors.forename: Carolina W
authors.initials: CW
authors.lastname: Ribeiro
authors.forename: Andrea
authors.initials: A
authors.lastname: Soares-Costa
authors.forename: Maria Cristina
authors.initials: MC
authors.lastname: Falco
authors.forename: Sabrina M
authors.initials: SM
authors.lastname: Chabregas
authors.forename: Eugênio C
authors.initials: EC
authors.lastname: Ulian
authors.forename: Simone S
authors.initials: SS
authors.lastname: Cotrin
authors.forename: Adriana K
authors.initials: AK
authors.lastname: Carmona
authors.forename: Lucimeire A
authors.initials: LA
authors.lastname: Santana
authors.forename: Maria Luiza V
authors.initials: ML
authors.lastname: Oliva
authors.forename: Flavio
authors.initials: F
authors.lastname: Henrique-Silva
mesh_terms: D015891:Cystatins / Q000235:genetics* / Q000302:isolation & purification*; D006639:Histidine / Q000737:chemistry / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics* / Q000302:isolation & purification*; D030821:Plants, Genetically Modified / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D025801:Ubiquitin / Q000235:genetics; D003313:Zea mays / Q000235:genetics
chemical_list: D015891:Cystatins; D010940:Plant Proteins; D011994:Recombinant Proteins; D025801:Ubiquitin; C466627:canecystatin protein, sugarcane; D006639:Histidine