Si-tag for silica particle based protein purification and oriented immobilization to silicon surface

>gi|281180352|dbj|BAI56682.1| 50S ribosomal protein L2 [Escherichia coli SE15]
MAVVKCKPTSPGRRHVVKVVNPELHKGKPFAPLLEKNSKSGGRNNNGRITTRHIGGGHKQAYRIVDFKRN
KDGIPAVVERLEYDPNRSANIALVLYKDGERRYILAPKGLKAGDQIQSGVDAAIKPGNTLPMRNIPVGST
VHNVEMKPGKGGQLARSAGTYVQIVARDGAYVTLRLRSGEMRKVEADCRATLGEVGNAEHMLRVLGKAGA
ARWRGVRPTVRGTAMNPVDHPHGGGEGRNFGKHPVTPWGVQTKGKKTRSNKRTDKFIVRRRSK

>gi|281177210:3409282-3410103 Escherichia coli SE15 DNA, complete genome
TTATTTGCTACGGCGACGTACGATGAATTTATCAGTACGCTTGTTGCTGCGGGTCTTCTTACCTTTGGTC
TGAACGCCCCACGGAGTTACCGGGTGCTTACCAAAGTTACGACCTTCACCACCACCATGTGGGTGGTCTA
CCGGGTTCATCGCGGTACCGCGAACGGTCGGACGAACACCACGCCAGCGTGCAGCACCTGCTTTACCCAG
AACGCGCAGCATATGCTCAGCATTGCCAACTTCGCCCAGAGTTGCACGGCAGTCTGCTTCTACTTTACGC
ATTTCACCAGAACGCAGACGCAGGGTGACATAAGCACCATCACGAGCAACGATCTGAACGTAAGTACCAG
CGGAACGTGCCAGCTGACCGCCTTTACCTGGTTTCATTTCTACGTTATGAACAGTAGAACCAACCGGGAT
GTTGCGCATCGGCAGGGTGTTACCTGGTTTGATTGCAGCATCAACGCCAGACTGAATCTGGTCGCCAGCT
TTCAGGCCTTTAGGGGCCAGGATGTAACGGCGTTCACCGTCTTTGTACAGAACCAGCGCGATGTTCGCGG
AACGGTTCGGATCGTACTCAAGACGTTCAACAACTGCCGGGATACCGTCTTTGTTGCGTTTGAAGTCAAC
AATACGGTAAGCCTGCTTGTGGCCACCACCGATATGACGAGTGGTGATACGGCCATTGTTGTTACGACCA
CCGGATTTGCTGTTTTTTTCCAGCAACGGAGCAAAAGGTTTGCCCTTGTGCAGCTCAGGGTTAACCACTT
TAACTACGTGGCGACGACCCGGAGATGTCGGTTTACATTTAACAACTGCCAT

This tag was mass spec from proteins bound to silica particles. Kd is 0.7 nM. The tag size is 273aa, not very big. A truncation version can also be used. Compare to purification using Hisx6 tag, Si-tag gives less pure but more protein. For me, the most interesting application of Si-tag is oriented immobilization of protein to silicon surface.

Glass was made of silia. Right?

Biotechnol Bioeng. 2007 Apr 15;96(6):1023-9.

The Si-tag for immobilizing proteins on a silica surface.

Taniguchi K, Nomura K, Hata Y, Nishimura T, Asami Y, Kuroda A.

Department of Molecular Biotechnology, Hiroshima University, Hiroshima 739-8530, Japan.

Targeting functional proteins to specific sites on a silicon device is essential for the development of new biosensors and supramolecular assemblies. Using intracellular lysates of several bacterial strains, we found that ribosomal protein L2 binds tightly to silicon particles, which have surfaces that are oxidized to silica. A fusion of E. coli L2 and green fluorescence protein adsorbed to the silica particles with a K(d) of 0.7 nM at pH 7.5 and also adsorbed to glass slides. This fusion protein was retained on the glass slide even after washing for 24 h with a buffer containing 1 M NaCl. We mapped the silica-binding domains of E. coli L2 to amino acids 1-60 and 203-273. These two regions seemed to cooperatively mediate the strong silica-binding characteristics of L2. A fusion of L2 and firefly luciferase also adsorbed on the glass slide. This L2 silica-binding tag, which we call the "Si-tag," can be used for one-step targeting of functional proteins on silica surfaces. (c) 2006 Wiley Periodicals, Inc.

PMID: 17013933

Protein Expr Purif. 2009 Dec 21. [Epub ahead of print]

Single-step affinity purification of recombinant proteins using the silica-binding Si-tag as a fusion partner.

Ikeda T, Ninomiya KI, Hirota R, Kuroda A.

Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8530, Japan; Research Institute for Nanodevice and Bio Systems, Hiroshima University, 1-4-2 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8527, Japan.

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. Copyright © 2009. Published by Elsevier Inc.

PMID: 20034569