Protein purification using combined streptavidin (or avidin)-Sepharose and thiopropyl-Sepharose affinity chromatography.
The major problem usually encountered in the application of the (strept)avidin-biotin system to the purification of proteins (or other biological molecules) lies in the difficult reversion of the interaction between immobilized (strept)avidin and the adsorbed biotinylated protein. Among the proposed solutions is the selective biotinylation of the entity to be purified by a disulphide-containing biotinylated reagent which allows its recovery from (strept)avidin gels by dithiothreitol (DTT) treatment. As emphasized by the example of angiotensin II receptor purification, achieved using this strategy, optimum reduction of this disulphide bridge may require improvement of its accessibility using denaturating agents such as sodium dodecyl sulphate or urea. However, these agents release important amounts of (strept)avidin. Two general ways of solving this problem are proposed. One solution takes advantage of the absence of cysteine in the streptavidin sequence: the protein to be purified is selectively readsorbed to thiopropyl-Sepharose through the thiol function generated on DTT cleavage of the biotinylated reagent. The other solution is an empirical approach to make possible the use of avidin, which possesses cysteine residues: combined avidin-Sepharose and thiopropyl-Sepharose chromatography proved efficient when carried out in the presence of urea as denaturing agent.
Desarnaud
F
F
Centre CNRS-INSERM de Pharmacologie-Endocrinologie, Montpellier, France.
Marie
J
J
Larguier
R
R
Lombard
C
C
Jard
S
S
Bonnafous
J C
JC
D000818:Animals; D001360:Avidin / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry; D002462:Cell Membrane / Q000737:chemistry; D002846:Chromatography, Affinity / Q000379:methods*; D004229:Dithiothreitol / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D008099:Liver / Q000737:chemistry; D051381:Rats; D011945:Receptors, Angiotensin / Q000302:isolation & purification*; D012685:Sepharose / Q000031:analogs & derivatives / Q000737:chemistry; D019809:Streptavidin
D001426:Bacterial Proteins; D011945:Receptors, Angiotensin; D001360:Avidin; C035872:thiopropyl-sepharose; D012685:Sepharose; D019809:Streptavidin; D004229:Dithiothreitol