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Publication Details
AFRICAN RESEARCH NEXUS
SHINING A SPOTLIGHT ON AFRICAN RESEARCH
biochemistry, genetics and molecular biology
Oriented immobilization of the tobacco etch virus protease for the cleavage of fusion proteins
Journal of Biotechnology, Volume 158, No. 3, Year 2012
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Description
The tobacco etch virus (TEV) protease is a useful tool for the removal of fusion tags from recombinant proteins. The difficulty in obtaining this enzyme led us to look for an optimal method for its use. In this work, we produced both the wild-type and the S219V mutant TEV proteases fused to the Streptag II affinity sequence (Streptag II-TEV WT, and Streptag II-TEV S219V, respectively). The two enzymes were affinity immobilized on a streptavidin-agarose matrix and compared to their respective free forms. Both immobilized Streptag II-TEV WT and Streptag II-TEV S219V were active on the 74-kDa Streptag II substrate with a retained activity of 83.5% and 81%, respectively compared to their free corresponding forms. The slight enzyme activity decrease caused by the immobilization was balanced by the enhanced stability and the successful repetitive use of the proteolytic columns. Thus, the wild-type and the mutant immobilized proteases were used, during a period of 18 months, for nine batch reactions with retention of 38% and 51% of their initial activities, respectively. The present results demonstrate that immobilized TEV protease on streptavidin-agarose is an attractive and efficient tool for fusion protein cleavage, especially when the target protein is fused to a streptagged fusion partner. Using this strategy, the total process can be shortened by performing the cleavage and the recovery of the purified target protein in one step. © 2012 Elsevier B.V.
Authors & Co-Authors
Miladi, Baligh
France, Cergy
Ecole de Biologie Industrielle Ebi
France, Neuville-sur-oise
Equipe de Recherche Sur Les Relations Matrice Extracellulaire-cellule
Marjou, Ahmed El
France, Paris
Biologie Cellulaire et Cancer
Boeuf, Guilhem
France, Cergy
Ecole de Biologie Industrielle Ebi
Bouallagui, Hassib
Tunisia, Tunis
University of Carthage
Dufour, Florence
France, Cergy
Ecole de Biologie Industrielle Ebi
Di Martino, Patrick
France, Neuville-sur-oise
Equipe de Recherche Sur Les Relations Matrice Extracellulaire-cellule
Elm'selmi, Abdelatif
France, Cergy
Ecole de Biologie Industrielle Ebi
Statistics
Citations: 32
Authors: 7
Affiliations: 4
Identifiers
Doi:
10.1016/j.jbiotec.2012.01.010
ISSN:
01681656
e-ISSN:
18734863
Research Areas
Substance Abuse