The biotin-streptavidin interaction can be reversibly broken using water at elevated temperatures.
The biotin-streptavidin system is the strongest noncovalent biological interaction known, having a dissociation constant, K(d), in the order of 4x10(-14) M. The strength and specificity of the interaction has led it to be one of the most widely used affinity pairs in molecular, immunological, and cellular assays. However, it has previously been impossible to re-use any streptavidin solid support, since the conditions needed to break the interaction with biotin has led to the denaturation of the streptavidin. Here, we show that a short incubation in nonionic aqueous solutions at temperatures above 70 degrees C can efficiently break the interaction without denaturing the streptavidin tetramer. Both biotin and the streptavidin remain active after dissociation and both molecules can therefore be re-used. The efficiency of the regeneration allowed solid supports with streptavidin to be used many times, here exemplified with the multiple re-use of streptavidin beads used for sample preparation prior to automated DNA sequencing. The results suggest that streptavidin regeneration can be introduced as an improvement in existing methods and assays based on the streptavidin system as well as emerging solid phase applications in fields, such as microfluidics and nanotechnology.
Holmberg
Anders
A
Department of Biotechnology, Royal Institute of Technology (KTH), Stockholm, Sweden.
Blomstergren
Anna
A
Nord
Olof
O
Lukacs
Morten
M
Lundeberg
Joakim
J
Uhlén
Mathias
M
D001322:Autoanalysis / Q000295:instrumentation; D001710:Biotin / Q000737:chemistry* / Q000302:isolation & purification*; D004247:DNA / Q000302:isolation & purification; D004587:Electrophoresis, Agar Gel / Q000379:methods; D019075:Electrophoresis, Capillary / Q000379:methods; D005434:Flow Cytometry; D006358:Hot Temperature; D008280:Magnetics; D008863:Microspheres; D009994:Osmolar Concentration; D010957:Plasmids / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods; D017422:Sequence Analysis, DNA / Q000379:methods*; D012996:Solutions; D019809:Streptavidin / Q000737:chemistry* / Q000302:isolation & purification*; D014867:Water / Q000737:chemistry*
D012996:Solutions; D014867:Water; D001710:Biotin; D004247:DNA; D019809:Streptavidin