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.