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.
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 microM) and bovine serum albumin (BSA) (0.39 microM), 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%.
Soh
Nobuaki
N
Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, 6-10-1 Hakozaki, Fukuoka 812-8581, Japan.
Umeno
Daisuke
D
Tang
Zhonglan
Z
Murata
Masaharu
M
Maeda
Mizuo
M
D000180:Acrylic Resins / Q000737:chemistry; D000345:Affinity Labels; D011232:Chemical Precipitation; D004247:DNA / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D016385:TATA Box; D035181:TATA-Box Binding Protein / Q000302:isolation & purification
D000180:Acrylic Resins; D000345:Affinity Labels; D004268:DNA-Binding Proteins; D035181:TATA-Box Binding Protein; C052970:poly-N-isopropylacrylamide; D004247:DNA