Production of a His-tagged canecystatin in transgenic sugarcane and subsequent purification.

Dept of Genetic and Evolution, Laboratory of Molecular Biology, Federal University of São Carlos, São Carlos, Brazil.

Biotechnology progress 2008 Sep-Oct

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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.

Citation

Carolina W Ribeiro, Andrea Soares-Costa, Maria Cristina Falco, Sabrina M Chabregas, Eugênio C Ulian, Simone S Cotrin, Adriana K Carmona, Lucimeire A Santana, Maria Luiza V Oliva, Flavio Henrique-Silva. Production of a His-tagged canecystatin in transgenic sugarcane and subsequent purification. Biotechnology progress. 2008 Sep-Oct;24(5):1060-6