Expression of Barley Leaf Cysteine Proteinase Inhibitor as an Active Fusion Protein in Escherichia coli Cells
Nowadays, identification of the novel physio-biological and therapeutic functions of plant cysteine proteinase inhibitors “plant cystatins / phytocystatins” are the great of interests for molecular biologists. Whether for biochemical, structural or functional studies, their successful expression along with an easy purification method is required. To date, fusion tags are the best available tools that meet all those requirements. We report here the cloning and simple functional expression and purification of a barley putative cystatin in Escherichia coli cells. For the first time, a part of barley coding sequence containing a predicted active cystatin was amplified by polymerase chain reaction and expressed as maltose binding fusion protein in TB1 strain of E. coli cells using pMALc2X over-expression vector system without affecting the bacterial growth. The expressed product was purified by single step affinity chromatography from the soluble fraction of induced culture at a yield of about 37 mg/ liter of bacterial cell culture. The purified fused protein could efficiently inhibit papain activity in vitro without the cleavage of the fusion partner.
Keywordsbarley cystatin proteinase inhibitor fusion protein expression
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- Botella, M.A., Xu, Y., Prabha, T.N., Zhao, Y., Narasimhan, M.L., Wilson, K.A., Nielsen, S.S., Bressan, R.A., Hasegawa, P.M. 1996. Differential expression of soybean cysteine proteinase inhibitor genes during development and in response to wounding and methyl jasmonate. Plant Physiol. 112:1201–1210.CrossRefGoogle Scholar
- Cipriani, G., Fuentes, S., Bello, V., Salazar, L.F., Ghislain, M., Zhang, D.P. 2000. Transgene expression of rice cysteine proteinase inhibitors for the development of resistance against sweetpotato feathery mottle virus. In: CIP (Centro Internacional de la Papa) Program Report 1999–2000. Lima, Peru, pp. 267–271.Google Scholar
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