Purifying the hedgehog protein and its variants.
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.
Baker
Darren P
DP
Taylor
Frederick R
FR
Pepinsky
R Blake
RB
D000215:Acylation; D000818:Animals; D002460:Cell Line; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D053823:Hedgehog Proteins / Q000737:chemistry / Q000302:isolation & purification*; D006801:Humans; D007313:Insecta; D051379:Mice; D008970:Molecular Weight; D050505:Mutant Proteins / Q000737:chemistry / Q000302:isolation & purification*; D010843:Pichia; D015202:Protein Engineering; D011499:Protein Processing, Post-Translational; D051381:Rats; D012995:Solubility
D053823:Hedgehog Proteins; D050505:Mutant Proteins