Single-step affinity purification of recombinant proteins using the silica-binding Si-tag as a fusion partner.
We previously reported that a silica-binding protein, designated Si-tag, can be used as a fusion tag to immobilize functional proteins on silica surfaces. In this study, by taking advantage of the strong affinity of Si-tag for silica, we developed a single-step purification method for Si-tagged fusion proteins. We utilized unmodified bare silica particles as a specific adsorbent and a high concentration of MgCl(2) solution as an elution buffer. A fusion protein of Si-tag and immunoglobulin-binding staphylococcal protein A, designated Si-tagged protein A, was recovered with a purity of 87+/-3% and yield of 84+/-4% from a crude extract of recombinant Escherichia coli. The simplicity of our method enables rapid, cost-effective purification of Si-tagged fusion proteins. We also discuss the mechanism of binding and dissociation of Si-tag and silica surfaces, and we suggest that the unusual basicity and disordered structure of the Si-tag polypeptide play important roles in the binding to silica.
Ikeda
Takeshi
T
Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8530, Japan. ikedatakeshi@hiroshima-u.ac.jp
Ninomiya
Ken-ichi
K
Hirota
Ryuichi
R
Kuroda
Akio
A
D000327:Adsorption; D001681:Biological Assay; D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006639:Histidine / Q000378:metabolism; D007074:Immunoglobulin G / Q000276:immunology; D009842:Oligopeptides / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification*; D012822:Silicon Dioxide / Q000378:metabolism*; D013205:Staphylococcal Protein A / Q000737:chemistry / Q000302:isolation & purification
C471213:His-His-His-His-His-His; D007074:Immunoglobulin G; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D006639:Histidine; D012822:Silicon Dioxide