Oriented immobilization of the tobacco etch virus protease for the cleavage of fusion proteins.
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.
Laboratoire de Biologie Moléculaire, Ecole de Biologie Industrielle, 32 Bd du Port, Cergy 95094, France.
Baligh
B
Miladi
Ahmed
A
El Marjou
Guilhem
G
Boeuf
Hassib
H
Bouallagui
Florence
F
Dufour
Patrick
P
Di Martino
Abdellatif
A
Elm'selmi
D010450:Endopeptidases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D010942:Plant Viruses / Q000201:enzymology* / Q000235:genetics; D059748:Proteolysis; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019809:Streptavidin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics
D004800:Enzymes, Immobilized; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D019809:Streptavidin; D010450:Endopeptidases; C099488:TEV protease