title: Characterization of the diatomite binding domain in the ribosomal protein L2 from E. coli and functions as an affinity tag.
abstract: The ribosomal protein L2, a constituent protein of the 50S large ribosomal subunit, can be used as Si-tag using silica particles for the immobilization and purification of recombinant proteins (Ikeda et al. (Protein Expr Purif 71:91-95, 2010); Taniguchi et al. (Biotechnol Bioeng 96:1023-1029, 2007)). We applied a diatomite powder, a sedimentary rock mainly composed with diatoms silica, as an affinity solid phase and small ubiquitin-like modifier (SUMO) technology to release a target protein from the solid phase. The L2 (203-273) was the sufficient region for the adsorption of ribosomal protein L2 on diatomite. We comparatively analyzed the different adsorption properties of the two deleted proteins of L2 (L2 (1-60, 203-273) and L2 (203-273)) on diatomite. The time required to reach adsorption equilibrium of L2 (203-273) fusion protein on diatomite was shorter than that of L2 (1-60, 203-273) fusion protein. The maximum adsorption capacity of L2 (203-273) fusion protein was larger than that of L2 (1-60, 203-273) fusion protein. In order to study whether the L2 (203-273) can function as an affinity purification tag, SUMO was introduced as one specific protease cleavage site between the target protein and the purification tags. The L2 (203-273) and SUMO fusion protein purification method was tested using enhanced green fluorescent protein as a model protein; the result shows that the purification performance of this affinity purification method was good. The strong adsorption characteristic of L2 (203-273) on diatomite also provides a potential protein fusion tag for the immobilization of enzyme.
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China.
authors.forename: Junhua
authors.initials: J
authors.lastname: Li
authors.forename: Yang
authors.initials: Y
authors.lastname: Zhang
authors.forename: Yanjun
authors.initials: Y
authors.lastname: Yang
mesh_terms: D000327:Adsorption; D001665:Binding Sites; D002846:Chromatography, Affinity / Q000379:methods; D007692:Diatomaceous Earth / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012269:Ribosomal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D025842:SUMO-1 Protein / Q000378:metabolism; D017384:Sequence Deletion
chemical_list: D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; D012269:Ribosomal Proteins; D025842:SUMO-1 Protein; C485184:enhanced green fluorescent protein; C560088:ribosomal protein L2, E coli; D049452:Green Fluorescent Proteins; D007692:Diatomaceous Earth; C033787:diatomite