Protein Expression and Purification 23, 440–446 (2001) doi:10.1006/prep.2001.1515, available online at http://www.idealibrary.com on One-Step Purification of Recombinant Proteins Using a Nanomolar-Affinity Streptavidin-Binding Peptide, the SBP-Tag Anthony D. Keefe, 1 David S. Wilson, 2 Burckhard Seelig, and Jack W. Szostak Department of Molecular Biology and Howard Hughes Medical Institute, Massachusetts General Hospital, Boston, Massachusetts 02114 Received May 7, 2001, and in revised form July 20, 2001 Protein affinity tags are widely used for the purifica- We describe the use of the SBP-tag, a new streptavi- tion and detection of recombinant proteins, particularly din-binding peptide, for both the one-step purification from complex mixtures such as lysed cells. However, and the detection of recombinant proteins. The SBP- only a small number of affinity tags are available, and tag sequence is 38 amino acids long and binds to strep- there are significant drawbacks associated with the use tavidin with an equilibrium dissociation constant of of many of them. Commonly used categories of tags, 2.5 nM. We demonstrate that a single-step purification and their limitations, are described below: 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 1. Fusion protein tags such as maltose-binding pro- of the immobilized streptavidin used to purify SBP- tein (MBP) 3 (1) and glutathione S-transferase (2), which tagged proteins is about 0.5 mg per milliliter of matrix, bind to maltose/amylose and glutathione, respectively. which is high enough to isolate large quantities of pro- These tags are suitable for large-scale protein purifica- teins for further study. Also, the elution conditions tion. However, these are large protein modules and as from the streptavidin column are very mild and spe- a result interfere with structural characterization of cific, consisting of the wash buffer plus biotin. This the resultant fusion protein. Also, proteins purified combination of high-affinity, high-yield, mild elution solely on the basis of these tags are not sufficiently pure conditions, and simplicity of use makes the SBP-tag suitable for high-throughput protein expression/ for some purposes. purification procedures, including robotically manip- 2. Antibody epitope-tags such as the myc-tag (3) and ulated protocols with microtiter plates. Additionally, the FLAG-tag (4) which bind to immobilized antibodies. the SBP-tag can be used for detection since a wide These tags are capable of higher degrees of protein variety of streptavidin-conjugated fluorescent and purification, but the yield of purified protein per volume enzymatic systems are commercially available. We also of matrix is low because antibodies are used as the present a new, rapid, method for the measurement of capture agents. protein–protein, protein–peptide, or protein–small molecule equilibrium dissociation constants that 3. The hexahistidine tag (His-tag) (5), which binds require as little as 1 fmol of labeled protein. We call to immobilized nickel and cobalt ions. The His-tag is this method the spin-filter binding inhibition assay. short and can be used to purify very large quantities of  2001 Academic Press His-tagged proteins. However, unless specific protocols 1 Current address: Archemix Inc., 20 Hampden Street, Boston, 3 Abbreviations used: MBP, maltose-binding protein; His-tag, hexa- histidine tag; DTT, dithiothreitol; NTA, nitrilotriacetic acid; LB, Luria MA 02119. 2 Current address: Zyomyx Inc., 3911 Trust Way, Hayward, CA broth; SBB, streptavidin-binding buffer; HRP, horseradish peroxi- dase; SBIA, spin-filter binding inhibition assay. 94545. 440 1046-5928/01 $35.00 Copyright  2001 by Academic Press All rights of reproduction in any form reserved.