Purifying the Hedgehog Protein and its Variants
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The purification of recombinant versions of the N-terminal signaling fragment of Sonic hedgehog (ShhN) from E. coli, Hi-5™ insect cells, yeast, and mammalian cell sources reveals diverse post-translational modifications that affect the potency of the purified protein. Modifications to the N-terminal cysteine with fatty acyl groups results in significant increases in potency, up to 100-fold, when compared with the unmodified protein. Proteolytic clipping at sites near the N-terminus results in inactivation of signaling activity. The ShhN protein is particularly sensitive to metal ion-induced oxidation, and the methods described here were developed to minimize this oxidation. The purification methods developed for ShhN were applicable to human Indian and Desert hedgehog N-terminal signaling proteins, and therefore should be useful for hedgehog proteins from other species.
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- Purifying the Hedgehog Protein and its Variants
- Book Title
- Hedgehog Signaling Protocols
- pp 1-22
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- Methods Inmolecular Biology™
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- Humana Press
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- Humana Press
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