Download PPT

On-column Chemical Refolding
Natalia Oganesyan and Rosalind Kim
Lawrence Berkeley National Laboratory
PPCW, March 29-31, 2004
The work described here was supported by the National
Institutes of Health GM 62412
Chemical Refolding
of protein inclusion bodies (IB)
Daugherty et al., 1998
JBC, vol. 273, 33961-71





IB in 8 M urea
Dilution into a large volume
of buffer with detergent
Dilution with -cyclodextrin
buffer
Filtration (0.2 μ)
Concentration
BSGC on-column refolding method for
non-membrane proteins





IB in 8 M urea
Binding to Ni-NTA
Wash with buffer containing
detergent
Wash with buffer containing
-cyclodextrin
Elution with imidazole
Time required
2-3 days
20 hrs
Detergent mini-refolding screen
IB in 8 M urea bound to Ni-NTA
CTAB
No detergent
DDM
OG
Triton X-100
Wash with -cyclodextrin on mini-columns
Elution
SDS-PAGE
Refolding with selected best detergent
BSGC refolding protocol








Solubilization of IB in 8 M urea or 6 M GuHCl
Overnight binding to Ni-NTA (Qiagen)
Column wash with urea buffer and 20 mM imidazole
Wash with 0.6 mM Triton X-100 or DDM under native
conditions
Wash with 5 mM -cyclodextrin
Wash with 0.5 M NaCl
Elution with 400 mM imidazole
IEX or SEC
Summary of refolded BSGC targets
Targets








MW, kDa
% “Refolded” *
1084B
40
1349B
20
1105B
70
1113B
19
1049B
36
1277B
44
1089B
16.7
3 targets failed to refold
40%
100%
100%
100%
50%
100%
100%
*% target eluted / % target loaded
Crystallized
yes
2.8 A data
3.2 A data/solved
yes
yes
yes
no
SeMet Crystals of 1105B
Refolded 1105B
0.1 M Tris-HCl, pH 8.5, 0.2 M Li2SO4,
30%(w/v) PEG 4K
Soluble 1105B
0.1 M HEPES, pH 7.5, 1.5 M Li2SO4
Crystal structure of 1105B
Refolded
3.2 Å P32
2 molecules per a.u.
Soluble
2.8 Å P43
6 molecules per a.u.
V.Oganesyan et al. Manuscript in preparation
Summary

On-column chemical refolding is fast and provides an
easy way to refold various His-tagged proteins.

Mini-purification screen allows selection of the most
efficient detergent. It can be formulated as a highthroughput method.

Seven out of ten tested proteins have been refolded
by this method.

Effective refolding provides enough soluble protein
for crystallization trials.