Eukaryotic Expression and Purification of Recombinant Extracellular Matrix Proteins Carrying the Strep II Tag - Methods in Molecular Biology: Extracellular Matrix Protocols - page 54

4. Expression of the protein as multiple bands when run on a SDS-PAGE gel may

have numerous causes.
a. Intra- or extracellular degradation of the protein may be occurring, the latter
can be reduced by protease inhibitors, more frequent harvesting of media from
the cells, or the use of different cell lines (e.g., COS cells) perhaps not
expressing the protease.
b. Variable levels of protein glycosylation may occur, this can be analyzed by
treatment of the protein with N-glycosidase F, which should give an
unglycosylated product of a single molecular mass if the differences are
caused by N-linked carbohydrates. This may be caused by very high expres-
sion of the protein leading to exhaustion of the post-translational mechanisms
of the cell. If it is a major hinderance, stable clones expressing at lower levels
may reduce these problems.

5. The protein is expressed, but does not bind when added to the StrepTactin column.

a. There has been insufficient cleaning of the column possibly owing to using
low volumes of the regeneration and washing buffers.
b. The medium was passed over the column at too great a speed for effi-
cient binding.
c. The tag is masked by folding of the protein, a tag placed on the opposite
terminal may be more effective.

Acknowledgements
The authors gratefully thank the BMBF in the framework of the ZMMK
(Centre for Molecular Medicine Cologne) and the Köln Fortune Fund of the
Medical Faculty, University of Cologne for their financial assistance.

References
1. Bork, P., Downing, A. K., Kieffer, B., and Campbell, I. D. (1996) Structure and
distribution of modules in extracellular proteins. Rev. Biophys . 29, 119-167.
2. Pöschl, E., Fox, J. W., Block, D., Mayer, U., and Timpl, R. (1994) Two noncon-
tiguous regions contribute to nidogen binding to a single EGF-like motif of the
laminin 1 chain. EMBO. J . 13, 3741-3747.

3. Kohfeldt, E., Maurer, P., Vannahme, C., and Timpl, R. (1997) Properties of the
extracellular calcium binding module of the proteoglycan testican. FEBS. Lett .
414, 557-561.
4. Kohfeldt, E., Gohring, W., Mayer, U., Zweckstetter, M., Holak, T. A., Chu, M.
L., and Timpl, R. (1996) Conversion of the Kunitz-type module of collagen VI
into a highly active trypsin inhibitor by site-directed mutagenesis. Eur. J. Biochem .
238, 333-340.
5. Schmidt, T. G. and Skerra, A. (1993) The random peptide library-assisted engi-
neering of a C-terminal affinity peptide, useful for the detection and purification
of a functional Ig Fv fragment. Protein Eng . 6, 109-122.
6. Schmidt, T. G., Koepke, J., Frank, R., and Skerra, A. (1996) Molecular interac-
tion between the Strep-tag affinity peptide and its cognate target, streptavidin. J.
Mol. Biol. 255, 753-766.

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