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 protein-ases, can be used for protection against insect attacks. A transformed sugarcane plant that presented high levels of HISCaneCPI-1 expression, was selected for the purification of this protein through affinity chromatography in a nickel column. This purified HISCaneCPI-1 was immunodetected using a polyclonal antibody, which was also able to detect the HISCaneCPI-1 in a crude extract from transgenic plant leaves. Assays of inhibitory activity performed with the purified HISCaneCPI-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. © 2008 American Institute of Chemical Engineers.