title: Site specific and reversible protein immobilization facilitated by a DNA binding fusion tag.
abstract: Due to the complexity and diversity of protein structures, site-specific protein immobilization has always been challenging. On the contrary, DNA immobilization is straightforward with well established chemical methods. Single-strand DNA binding protein (SSB) binds tightly to single-stranded DNA (ssDNA). Herein, we investigated the feasibility of using SSB as a fusion tag to facilitate site-specific and reversible immobilization of target proteins. As a model system, we constructed a fusion protein by joining a superfolder green fluorescent protein (sfGFP) with SSB. The fluorescence emission and ssDNA binding affinity of the fusion protein were compared separately with those of the individual modules. Both modules fully retained their properties in the fusion construct. Next, we covalently attached ssDNA (dT(37)) to a supporting matrix through either amine or thiol functionalization. The attached ssDNA mediated reversible sfGFP-SSB immobilization. The immobilized protein could be released through changes of conditions including pH, concentration of divalent cations, and the presence of the complementary dA(35) oligonucleotide.
authors.affiliation: Department of Chemistry, University of Kentucky, Lexington, Kentucky 40506-0055, USA.
authors.forename: Meng
authors.initials: M
authors.lastname: Zhong
authors.forename: Jun
authors.initials: J
authors.lastname: Fang
authors.forename: Yinan
authors.initials: Y
authors.lastname: Wei
mesh_terms: D003001:Cloning, Molecular; D004277:DNA, Single-Stranded / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000302:isolation & purification; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008954:Models, Biological; D009841:Oligonucleotides / Q000737:chemistry; D010316:Particle Size; D013050:Spectrometry, Fluorescence; D013499:Surface Properties
chemical_list: D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D009841:Oligonucleotides; D049452:Green Fluorescent Proteins