Analytical Sciences

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, and Mizuo MAEDA 

Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, 6-10-1 Hakozaki, Fukuoka 812-8581, Japan

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 uM) and bovine serum albumin (BSA) (0.39 uM), 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%.

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