| Dilution |
| No washing step between solubilization and refolding |
| 8 M urea and 50 mM glycine (pH 8.0) |
| 328 mM ethanolamine and 0.5 % n-dodecyl-N,N-dimethyl-1-ammonio-3-propanesulfonate (=SB-12) (pH12) |
| None |
| no tag |
| 12.0 |
| 4.0 |
| 0.1 mg/ml |
|
| None |
| n/a |
| Inclusion bodies were collected by a low speed centrifugation step and solubilized in 8 M urea (see solubilization buffer). 10 mg/ml solubilzed protein was diluted 100-fold in refolding buffer containing 0.5 % (purified!) SB12 (see refolding buffer)which is approximately 5 x the critical micelle concentration. Important to note is that SB12 which can be purchased from Fluka (Buchs, Switzerland) needs to be purified by passing a solution of detergent in methanol/chloroform (1:1) over an Al2O3 column to remove all acidic impurities present in the commercial preparation.
After incubation overnight at 4 C, the refolding buffer (con taining refolded protein) was neutralised with HCl to pH7.5 and 10 mM Tris was added to buffer the solution. This neutralization was performed to stabilize the refolded state of the protein (conformation of protein unstable in alkaline conditions). Subsequently, to remove ethanolamine the solution was washed in a concentrator with 10 mM Tris-HCl, 0.5 % SB12, pH 7.5 (buffer A).
To remove unfolded protein and other contaminants, the in vitro folded Opa protein was purified by ion-exchange chromatography. An SP-Sepharose-HP column was equilibrated with buffer A, loaded with approximately 10 mg refolded protein and washed twice. The protein was eluted with a linear gradient of NaCl from 0 to 1 M. Because the Opa proteins belong to the group of heat-modifiable proteins, refolding could easily be monitored by SDS-PAGE performed under semi-native and denaturing conditions at the same time this allowed us to check the purity of the protein. Refolding was confirmed by far- and near-UV circular dichroism analyses.
|
| SDS-PAGE |
| None |
| None |
| NULL |
| > 95 % |
|
| Complete amino acid sequence of OpaJ129 (wild-type):
ASEDGSRSPYYVQADLAYAAERITHDYPQATGANNTSTVSDYFRNIRAHSIHPRVSVGYDFGGWRIAADY
ASYRKWKESNSSTKKVTEEINNNYKETQTKHQGNGSFHASSSLGLSAIYDFKLNDKFKPYIGARVAYGHV
KHQVHSVETKTTIVTSKPTQGAAQGGPIIQTDPSKPPYHESHSISSVGLGVIAGVGFDITPKLTLDTGYR
YHNWGRLENTRFKTHEVSLGMRYRF
|