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5
Eukaryotic Expression and Purification
of Recombinant Extracellular Matrix Proteins
Carrying the Strep II Tag
Neil Smyth, Uwe Odenthal, Barbara Merkl, and Mats Paulsson
1. Introduction
For recombinant expression of extracellular matrix (ECM) proteins or their
individual domains, the use of transformed mammalian cells offers two major
advantages. First, eukaryotic expression can be expected under optimum con-
ditions to produce a large proportion of correctly folded molecules. ECM pro-
teins are made from a group of 25 structurally known
(1)
and about 200 cDNA
derived domains many of which regularly reappear in the different proteins.
These have often a complex secondary structure, maintained by multiple disul-
fide bonds. Whereas by denaturing and then carefully renaturing, an approxi-
mation to the native structure may be obtained using prokaryotic expression
systems, the best that may be expected is that a small percentage of the protein
folds into such a conformation. Second, most ECM proteins are at least to some
extent glycosylated and often heavily so, and the use of the mammalian system
offers the best approximation to the sugar structures present in the native form
of the molecule.
While the use of the mammalian systems has the above advantages, it also
has major drawbacks. They are relatively slow to set up as opposed to those
using yeast, bacteria, or baculovirus/insect cells. It takes up 2 wk to obtain 1 L
of culture medium from mammalian cells, as opposed to 8 to 12 h from bacte-
ria. Tissue culture over such periods is labor intensive and expensive. Finally,
the level of the secreted protein in the media is variable (50
g-3 mg per L of
medium) and where no antibody exists to the protein or domain, this can lead
to problems in its identification and in following its progress during subse-
quent purification steps. A tag fused either on the N' or C' terminus can act as
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