A new tagging system for production of recombinant proteins in Drosophila S2 cells using the third domain of the urokinase receptor.
The use of protein fusion tag technology greatly facilitates detection, expression and purification of recombinant proteins, and the demands for new and more effective systems are therefore expanding. We have used a soluble truncated form of the third domain of the urokinase receptor as a convenient C-terminal fusion partner for various recombinant extracellular human proteins used in basic cancer research. The stability of this cystein-rich domain, which structure adopts a three-finger fold, provides an important asset for its applicability as a fusion tag for expression of recombinant proteins. Up to 20mg of intact fusion protein were expressed by stably transfected Drosophila S2 cells per liter of culture using this strategy. Purification of these secreted fusion proteins from the conditioned serum free medium of S2 cells was accompanied by an efficient one-step immunoaffinity chromatography procedure using the immobilized anti-uPAR monoclonal antibody R2. An optional enterokinase cleavage site is included between the various recombinant proteins and the linker region of the tag, which enables generation of highly pure preparations of tag-free recombinant proteins. Using this system we successfully produced soluble and intact recombinant forms of extracellular proteins such as CD59, C4.4A and vitronectin, as well as a number of truncated domain constructs of these proteins. In conclusion, the present tagging system offers a convenient general method for the robust expression and efficient purification of a variety of recombinant proteins.
Gårdsvoll
Henrik
H
Finsen Laboratory, Rigshospitalet, Strandboulevarden 49, DK-2100, Copenhagen Ø, Denmark. gvoll@finsenlab.dk
Hansen
Line V
LV
Jørgensen
Thomas J D
TJ
Ploug
Michael
M
D000818:Animals; D002478:Cells, Cultured; D004330:Drosophila / Q000166:cytology*; D015870:Gene Expression / Q000502:physiology*; D006801:Humans; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D055293:Receptors, Urokinase Plasminogen Activator; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
C524898:PLAUR protein, human; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D011994:Recombinant Proteins