We describe the use of the SBP-tag, a new streptavidin-binding peptide, for both the one-step purification and the detection of recombinant proteins. The SBP-tag sequence is 38 amino acids long and binds to streptavidin with an equilibrium dissociation constant of 2.5 nM. We demonstrate that a single-step purification of SBP-tagged proteins from bacterial extract yields samples that are more pure than those purified using maltose-binding protein or the His-tag. The capacity of the immobilized streptavidin used to purify SBP-tagged proteins is about 0.5 mg per milliliter of matrix, which is high enough to isolate large quantities of proteins for further study. Also, the elution conditions from the streptavidin column are very mild and specific, consisting of the wash buffer plus biotin. This combination of high-affinity, high-yield, mild elution conditions, and simplicity of use makes the SBP-tag suitable for high-throughput protein expression/purification procedures, including robotically manipulated protocols with microtiter plates. Additionally, the SBP-tag can be used for detection since a wide variety of streptavidin-conjugated fluorescent and enzymatic systems are commercially available. We also present a new, rapid, method for the measurement of protein-protein, protein-peptide, or protein-small molecule equilibrium dissociation constants that require as little as 1 fmol of labeled protein. We call this method the spin-filter binding inhibition assay.
Bibliographical noteFunding Information:
We thank members of the Szostak lab, especially Glenn Short and Glen Cho, for advice and Pamela Svec for minipreps. J.W.S. is an investigator of the Howard Hughes Medical Institute; additional funding was provided by the Cancer Research Fund of the Damon Runyon±Walter Winchell Foundation, the Emmy Noether Program of the Deutsche Forschungsgesellschaft, the NASA Astrobiology Institute, and the NIH. The GenBank accession number of pTAG2K with an insert encoding amino acids 22 to 67 of clone 18±19 from (16) (the multiply-tagged protein) is AY033554.