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

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• 作者：Noah Malmstadt ; David E. Hyre ; Zhongli Ding ; Allan S. Hoffman ; and Patrick S. Stayton
• 刊名：Bioconjugate Chemistry
• 出版年：2003
• 出版时间：May 2003
• 年：2003
• 卷：14
• 期：3
• 页码：575 - 580
• 全文大小：105K
• 年卷期：v.14,no.3(May 2003)
• ISSN：1520-4812

文摘

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 isshown to coprecipitate biotinylated immunoglobulin G (IgG) and a biotinylated oligonucleotide inresponse to a thermal stimulus. Thermally precipitated biotinylated macromolecules can be releasedfrom the S45A-PNIPAAm conjugate by simple treatment with excess free biotin. This release stephas 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 excessfree 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 targetmolecule (near 100%), but significantly smaller for the biotinylated oligonucleotide target (approximately 60% for the S45A-PNIPAAm conjugate compared to 80% for the WT-PNIPAAmconjugate). The release efficiency (fraction of originally precipitated target molecule released aftertreatment with free biotin) of the S45A-PNIPAAm conjugate is 70-80% for the biotinylated IgGtarget and nears 100% for the biotinylated oligonucleotide target. This system demonstrates the useof a high off-rate streptavidin mutant to add reversibility to a system based on smart-polymer-streptavidin conjugates.