Production of a His-tagged canecystatin in transgenic sugarcane and subsequent purification.
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.
Ribeiro
Carolina W
CW
Dept of Genetic and Evolution, Laboratory of Molecular Biology, Federal University of São Carlos, São Carlos, Brazil.
Soares-Costa
Andrea
A
Falco
Maria Cristina
MC
Chabregas
Sabrina M
SM
Ulian
Eugênio C
EC
Cotrin
Simone S
SS
Carmona
Adriana K
AK
Santana
Lucimeire A
LA
Oliva
Maria Luiza V
ML
Henrique-Silva
Flavio
F
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
D015891:Cystatins; D010940:Plant Proteins; D011994:Recombinant Proteins; D025801:Ubiquitin; C466627:canecystatin protein, sugarcane; D006639:Histidine