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Production of a His-tagged canecystatin in transgenic sugarcane and subsequent purification
- Source :
- Biotechnology progress. 24(5)
- Publication Year :
- 2009
-
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.
- Subjects :
- biology
Reverse Transcriptase Polymerase Chain Reaction
Ubiquitin
Transgene
fungi
Genetically modified crops
Plants, Genetically Modified
Cystatins
Zea mays
Recombinant Proteins
law.invention
Cathepsin L
Affinity chromatography
Biochemistry
law
Polyclonal antibodies
biology.protein
Recombinant DNA
Histidine
Cystatin
Promoter Regions, Genetic
Biotechnology
Cysteine
Plant Proteins
Subjects
Details
- ISSN :
- 15206033
- Volume :
- 24
- Issue :
- 5
- Database :
- OpenAIRE
- Journal :
- Biotechnology progress
- Accession number :
- edsair.doi.dedup.....09370742a3c3a5c3ef26324d574699a2