Biochemical characterization of human Upf1 helicase.
We present here the biochemical characterization of human Upf1 helicase core (hUpf1c). hUpf1c is overexpressed as a GST fusion protein in Escherichia coli and purified using chromatographic methods. In vitro ATP binding and single-stranded RNA (ssRNA) binding activities are measured using dot-blot technique. Measurement of RNA-dependent ATPase activity is performed by thin layer chromatography (TLC). The ATP-modulated ssRNA binding activity is examined by surface plasma resonance (SPR). The binding of double-stranded DNA (dsDNA) to hUpf1c is checked by electrophoretic mobility shift assay (EMSA, gel shift assay).
Cheng
Zhihong
Z
Cancer and Developmental Cell Biology Division, Institute of Molecular and Cell Biology, A STAR (Agency for Science, Technology and Research), Singapore, Singapore.
Morisawa
Gaku
G
Song
Haiwei
H
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002845:Chromatography / Q000379:methods; D006801:Humans; D009711:Nucleotides / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D020365:RNA Helicases; D020871:RNA Stability; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*
D009711:Nucleotides; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D012313:RNA; D000255:Adenosine Triphosphate; D020365:RNA Helicases; C101828:UPF1 protein, human