Oxidative refolding chromatography: folding of the scorpion toxin Cn5.
We have made an immobilized and reusable molecular chaperone system for oxidative refolding chromatography. Its three components-GroEL minichaperone (191-345), which can prevent protein aggregation; DsbA, which catalyzes the shuffling and oxidative formation of disulfide bonds; and peptidyl-prolyl isomerase-were immobilized on an agarose gel. The gel was applied to the refolding of denatured and reduced scorpion toxin Cn5. The 66-residue toxin, which has four disulfide bridges and a cis peptidyl-proline bond, had not previously been refolded in reasonable yield. We recovered an 87% yield of protein with 100% biological activity.
Cambridge Centre for Protein Engineering and Cambridge University Chemical Laboratory, MRC Centre, UK.
M M
MM
Altamirano
C
C
García
L D
LD
Possani
A R
AR
Fersht
D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods*; D002942:Circular Dichroism; D003445:Crustacea / Q000187:drug effects; D007928:Lethal Dose 50; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D017510:Protein Folding; D012604:Scorpion Venoms / Q000737:chemistry* / Q000633:toxicity; D013056:Spectrophotometry, Ultraviolet
D012604:Scorpion Venoms; C105894:crustacean-specific toxin 5, Centruroides