One-step purification of recombinant proteins using a nanomolar-affinity streptavidin-binding peptide, the SBP-Tag.
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.
Keefe
A D
AD
Department of Molecular Biology, Howard Hughes Medical Institute, Boston, Massachusetts 02114, USA.
Wilson
D S
DS
Seelig
B
B
Szostak
J W
JW
D000345:Affinity Labels / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002614:Chelating Agents / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D008320:Maltose / Q000378:metabolism; D008969:Molecular Sequence Data; D036103:Nanotechnology; D011485:Protein Binding; D014176:Protein Biosynthesis; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D012680:Sensitivity and Specificity; D019809:Streptavidin / Q000378:metabolism*; D014170:Transformation, Genetic
D000345:Affinity Labels; D002352:Carrier Proteins; D002614:Chelating Agents; D011993:Recombinant Fusion Proteins; C452700:streptavidin-binding peptide; D008320:Maltose; D019809:Streptavidin