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Refolding by dialysis

If the given rapid dilution protocol does not work out, reduce the final protein concentration (e.g., double refolding buffer to 200 volumes). Make sure your unfolded protein is pure (>90 %). Alternatively, refolding by dialysis can be tried [30, 31], however, we obtained our best result with the described rapid dilution procedure. If the protocol is transferred to other proteins, optimization of refolding work-up might be necessary. We recommend screening various concentrations and ratios of salts, supplements, pH values, and redox systems in analytical scale, similar as described in [37]. [Pg.117]

Figure 5. Percent of enzymatic activity recovered after renaturation by dialysis at 23 C. The protein was unfolded in 2.0 M GuHCl in 50 mM potassium phosphate buffer, pH 7.0 in the presence ( ) and absence ( a ) of 5 mM DTT. It was subsequently refolded in 50 mM potassium phosphate buffer, pH 6.0. The reduced enzyme was also dialyzed against 50 mM potassium phosphate, 5 mM DTT, pH 6.0 (o ) The final GuHCl concentration was 0.02 M. Figure 5. Percent of enzymatic activity recovered after renaturation by dialysis at 23 C. The protein was unfolded in 2.0 M GuHCl in 50 mM potassium phosphate buffer, pH 7.0 in the presence ( ) and absence ( a ) of 5 mM DTT. It was subsequently refolded in 50 mM potassium phosphate buffer, pH 6.0. The reduced enzyme was also dialyzed against 50 mM potassium phosphate, 5 mM DTT, pH 6.0 (o ) The final GuHCl concentration was 0.02 M.
The classic experiments conducted by Anfinsen and co-workers proved that all the information for the three-dimensional structure of a protein is encoded by its amino acid sequence (Anfinsen, 1973) Bovine pancreatic RNase A, a 124-residue protein that contains four disulfide bonds in its native state, was used as a model protein (Sela et al., 1959 White, 1961). RNase A denatures readily and its disulfide bonds are reduced by incubation in urea and 2-mercaptoethanol. After removal of urea and 2-mercaptoethanol by dialysis, a very slow but nearly complete recovery of catalytic activity is observed. Thus, Anfinsen concluded that it is possible to refold denatured proteins into their active state in the test tube. Based on this observation, he further noted that the information for the assumption of the native secondary and tertiary structure is contained in the amino acid sequence itself (Anfinsen et al., 1961). [Pg.283]

Many proteins that are completely unfolded In 8 M urea and p-mercaptoethanol (which reduces disulfide bonds) spontaneously renature (refold) Into their native states when the denaturing reagents are removed by dialysis. Because no cofactors... [Pg.68]

Although the titration curve of the guanidinated derivative in KCl (Fig. 148) does not show the three extra carboxyl groups, these groups appear when the titration is carried out in G. The titration curve of this derivative in KCl is almost identical, on, the acid side, with the KCl titration curve of the G-treated lysozyme, prepared by allowing native lysozyme to stand a few hours in 8 molar G and then removing the G by dialysis (see curve D of Fig. 143). These titration curves presumably differ from the titration cur eof the native material because of the different manner in which the molecule has refolded, after it was unfolded either by G or the conditions under which this derivative was prepared. [Pg.262]

Ke et al., 2004) and the signal recognition particle (SRP) M domain—4.5 S RNA complex (Batey et al., 2001) required complete denaturation in 8 M urea and refolding into the native form by slow dialysis to native conditions. Some RNAs in our experience have required extensive screening of ionic conditions, temperature protocols, and use of denaturants to find conditions that yield a near-homogeneous population. [Pg.123]

Methods presently employed for obtaining correctly refolded proteins from inclusion body preparations are often all-or-none propositions. They typically consist of denaturant solubilization, in urea or guanidine, followed by dilution or dialysis (2). Recovery of native activity or structure may be aided by using additives (enzyme inhibitors, co-factors, oxidation-reduction couples, etc.), which act to stabilize the native-state protein conformation. However, because such efforts are time-consuming and tedious, systematic examinations of solution conditions for protein folding/unfolding are rarely performed. [Pg.459]


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