Thermoprecipitation of Streptavidin via Oligonucleotide-Mediated Self-Assembly with Poly(N-isopropylacrylamide)
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- 作者：Robin B. Fong ; Zhongli Ding ; Cynthia J. Long ; Allan S. Hoffman ; and Patrick S. Stayton
- 刊名：Bioconjugate Chemistry
- 出版年：1999
- 出版时间：September 1999
- 年：1999
- 卷：10
- 期：5
- 页码：720 - 725
- 全文大小：105K
- 年卷期：v.10,no.5(September 1999)
- ISSN：1520-4812
文摘
A versatile strategy has been developed for selectively and sequentially isolating targets in a liquid-phase affinity separation environment. The strategy uses a recently developed approach for joiningtogether molecules in linkages that are defined by the complementary pairing of oligonucleotidesconjugated to the different molecules [Niemeyer, C. M., Sano, T., Smith, C. L., and Cantor, C. R.(1994) Nucleic Acids Res. 22, 5530-9]. In the work presented here, streptavidin was noncovalentlycoupled with the temperature-responsive poly(N-isopropylacrylamide) [poly(NIPAAM)] through thesequence-specific hybridization of oligonucleotides conjugated to the protein and polymer. A 20-meroligonucleotide was covalently linked through a heterobifunctional linker to a genetically engineeredstreptavidin variant that contained a unique cysteine residue at the solvent-accessible site Glu 116.The complementary DNA sequence was conjugated to the end of a linear ester-activated poly(NIPAAM).The two conjugates were allowed to self-assemble in solution via hybridization of their complementaryDNA sequences. The streptavidin-poly(NIPAAM) complex could be used to affinity-precipitateradiolabeled biotin or biotinylated alkaline phosphatase above 32 
C through the thermally inducedphase separation activity of the poly(NIPAAM). The streptavidin-oligo species could then be reversiblyseparated from the precipitated polymer-oligo conjugate and recycled by lowering the salt concentration, which results in denaturation of the short double-stranded DNA connection. The use ofoligonucleotides to couple polymer to streptavidin allows for selective precipitation of different polymersand streptavidin complexes based on the sequence-specific hybridization of their oligonucleotideappendages.
C through the thermally inducedphase separation activity of the poly(NIPAAM). The streptavidin-oligo species could then be reversiblyseparated from the precipitated polymer-oligo conjugate and recycled by lowering the salt concentration, which results in denaturation of the short double-stranded DNA connection. The use ofoligonucleotides to couple polymer to streptavidin allows for selective precipitation of different polymersand streptavidin complexes based on the sequence-specific hybridization of their oligonucleotideappendages.