Affinity precipitation separation of DNA binding protein using block conjugate composed of poly(N-isopropylacrylamide) grafted double-stranded DNA and double-stranded DNA containing a target sequence

Nobuaki Soh, Daisuke Umeno, Zhonglan Tang, Masaharu Murata, Mizuo Maeda

Research output: Contribution to journalArticlepeer-review

17 Citations (Scopus)

Abstract

In this research, we synthesized a novel DNA-polymer conjugate and evaluated its application to an affinity precipitation separation of TATA-box binding protein (TBP), which is a representative general transcription factor. The conjugate was composed of two fractions. One was a double-stranded DNA modified by the grafting of poly(N-isopropylacrylamide) (PNIPAAm), which is known as a thermosensitive vinyl polymer. The other fraction is a native double-stranded DNA containing a specific base sequence (5′-TATAAA-3′) called a TATA-box. These two fractions, which have EcoRI termini, were treated with T4 DNA ligase, and the block conjugate was obtained as a precipitate after two wash processes. When the resultant block conjugate was introduced into a sample solution containing TBP (0.26 μM) and bovine serum albumin (BSA) (0.39 μM), a rapid and selective precipitation separation of TBP under homogeneous conditions was achieved by controlling temperature. The purity of TBP in the precipitation fraction was estimated to be above 90%.

Original languageEnglish
Pages (from-to)1295-1299
Number of pages5
Journalanalytical sciences
Volume18
Issue number12
DOIs
Publication statusPublished - Dec 1 2002

All Science Journal Classification (ASJC) codes

  • Analytical Chemistry

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