Affinity Thermoprecipitation and Recovery of Biotinylated Biomolecules via a Mutant Streptavidin−Smart Polymer Conjugate

A system has been developed for reversibly binding and thermoprecipitating biotinylated macromolecules. A high off-rate Ser45Ala (S45A) streptavidin mutant has been covalently conjugated to poly(N-isopropylacrylamide) (PNIPAAm), a temperature-responsive polymer. The resulting conjugate is shown to coprecipitate biotinylated immunoglobulin G (IgG) and a biotinylated oligonucleotide in response to a thermal stimulus. Thermally precipitated biotinylated macromolecules can be released from the S45A-PNIPAAm conjugate by simple treatment with excess free biotin. This release step has been shown to be unique to the mutant streptavidin conjugate-a conjugate of wild type (WT) streptavidin and PNIPAAm does not release bound biotinylated molecules upon treatment with excess free biotin. The capture efficiency (fraction of target molecule precipitated from solution) of the S45A-PNIPAAm conjugate is similar to that of the WT-PNIPAAm conjugate for the biotinylated IgG target molecule (near 100%), but significantly smaller for the biotinylated oligonucleotide target (approximately 60% for the S45A-PNIPAAm conjugate compared to 80% for the WT-PNIPAAm conjugate). The release efficiency (fraction of originally precipitated target molecule released after treatment with free biotin) of the S45A-PNIPAAm conjugate is 70-80% for the biotinylated IgG target and nears 100% for the biotinylated oligonucleotide target. This system demonstrates the use of a high off-rate streptavidin mutant to add reversibility to a system based on smart-polymer-streptavidin conjugates.