Purification of membrane proteins in SDS and subsequent renaturation.
A prerequisite for the purification of any protein to homogeneity is that the protein is not non-specifically associated with other proteins especially during the final stage(s) of the fractionation procedure. This requirement is not so often fulfilled when nonionic detergents (for instance Triton X-100) are used for solubilization of membrane proteins. The reason is that these detergents are not efficient enough to prevent the protein of interest from forming aggregates with other proteins upon contact with chromatographic or electrophoretic supporting media, which, due to their polymeric nature, have a tendency to induce aggregation of other polymers, for instance, hydrophobic proteins. The aggregation can be avoided if sodium dodecyl sulfate (SDS) is employed as detergent. We therefore suggest that membrane proteins should be purified by conventional methods in the presence of SDS and that the purified proteins, which are in a denatured state, are allowed to renature. There is good change to renature internal membrane proteins since they should not be so susceptible to denaturation by detergents as are water-soluble proteins because the natural milieu of the former proteins is lipids which in fact are detergents. In this paper we present a renaturation method based on the removal of SDS by addition of a large excess of G 3707, a nonionic detergent. By this technique we have renatured a 5'-nucleotidase from Acholeplasma laidlawii and a neuraminidase from influenza virus. The enzyme activities were higher (up to 6-fold) after the removal of SDS than prior to the addition of SDS.
Hjertén
S
S
Institute of Biochemistry, University of Uppsala, Sweden.
Sparrman
M
M
Liao
J
J
D015720:5'-Nucleotidase; D000128:Acholeplasma laidlawii / Q000032:analysis* / Q000648:ultrastructure; D001426:Bacterial Proteins / Q000302:isolation & purification*; D002458:Cell Fractionation; D002462:Cell Membrane / Q000032:analysis / Q000648:ultrastructure; D008565:Membrane Proteins / Q000302:isolation & purification*; D009439:Neuraminidase / Q000032:analysis; D009708:Nucleotidases / Q000032:analysis; D009975:Orthomyxoviridae / Q000032:analysis*; D011489:Protein Denaturation; D012967:Sodium Dodecyl Sulfate; D014764:Viral Proteins / Q000302:isolation & purification*
D001426:Bacterial Proteins; D008565:Membrane Proteins; D014764:Viral Proteins; D012967:Sodium Dodecyl Sulfate; D009708:Nucleotidases; D015720:5'-Nucleotidase; D009439:Neuraminidase