Biology Reference
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an aid in the immunological identification of the expressed protein. It may
also be used in a variety of affinity chromotography methods leading to its
purification.
We use a modified version of the system described previously for expres-
sion of a variety of matrix proteins in human embryonic kidney 293 cells
(2-4) . The human cytomegalovirus immediate early gene enhancer-promoter
is used to drive the expression of the recombinant protein. This gives poten-
tially high expression of the product in primate cells, however, levels in rodent
lines may be minimal. The BM40 (SPARC/osteonectin) signal peptide is placed
downstream of the promoter and fused in frame to the protein with the tag. The
plasmid contains the cassette for the puromycin N-acetyltransferase gene
allowing selection in transfected eukaryotic cells and also an ampicillin resis-
tance gene for use in selection and plasmid manipulation in Escherichia coli.
Further, these plasmids also contain the Epstein Barr virus (EBV) origin of
replication. This is sensitive to the action of the EBV nuclear antigen, which is
expressed in 293-EBNA cells leading to the maintenance of the plasmid
extrachromosomally, with it replicating independently of the host cells divi-
sion. Hence, such plasmids can be used either to give stably integrated clones
or to produce cells with possibly higher protein production by expression of
the protein coded on the episomal plasmid. By maintaining the selection pres-
sure from the puromycin, these episomal plasmids are retained for prolonged
periods in such cells. There is, however, the possibility that with time the plas-
mid may be lost leading to a progressive reduction in the yield of recombinant
protein, though we have failed to see this even over 30 cell passages.
Many fusion tags have been successfully used to aid in the purification of
recombinant proteins. The tag peptide is a small sequence, usually 6-10 amino
acids long, which acts either as an antibody epitope or as an affinity ligand or
both. The first problem encountered with the use of a tag is that its activity
depends either upon it being immunogenic or participating in some other form
of molecular interaction. As generally the production of recombinant proteins
is to derive antisera or monoclonal antibodies or to study interactions, this may
be a handicap. This problem may be overcome by the addition of a protease
cleavage site between the tag and the protein to allow the removal of the tag
( see Note 1 ). Second, the position of the tag may affect the activity of the
expressed protein by altering folding or other post-translational modifications.
Finally, the tag itself may be masked by the tertiary structure of the protein.
Hence, care should be taken in deciding whether the tag should be placed to the
N- or C-terminus. We have successfully used the Strep II tag to give a single
step purification of recombinant proteins from the media of 293-EBNA cells.
This tag was initially found by screening a peptide library with streptavidin
and is highly specific, not binding to the closely related protein avidin (5) .
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