The Nano-tag, a streptavidin-binding peptide for the purification and detection of recombinant proteins.
We present a new streptavidin-binding peptide for both the purification and the detection of recombinant proteins. The peptide possesses nanomolar-affinity for streptavidin and therefore was termed Nano-tag. The Nano-tag(15) is 15 amino acids long and binds to streptavidin with a dissociation constant of 4 nM and the Nano-tag(9) is a 9-mer peptide with a dissociation constant of 17 nM. We demonstrate the one-step purification of Nano-tagged proteins, namely bovine heart fatty acid-binding protein (FABP), bacterial chloramphenicol acetyltransferase (CAT), and green fluorescent protein (GFP), from an in vitro translation system as well as from an Escherichia coli lysate. No significant influence of the Nano-tag(15) and of the conditions during affinity chromatography on maturation or activity of the proteins was observed whereas the Nano-tag(9) revealed a slight decline in the amount and activity of the synthesized proteins. The main advantage of the Nano-tag is the mild and specific elution with washing buffer plus biotin or related compounds, which enables the elution of the bound fusion protein from the streptavidin column in the native state. Additionally, the Nano-tag allowed the detection of recombinant proteins on Western blots by a streptavidin-alkaline phosphatase conjugate.
Lamla
Thorsten
T
Institut für Biochemie, Freie Universität Berlin, Thielallee 63, D-14195 Berlin, Germany.
Erdmann
Volker A
VA
D000345:Affinity Labels / Q000737:chemistry* / Q000378:metabolism; D000469:Alkaline Phosphatase / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002417:Cattle; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000302:isolation & purification; D002846:Chromatography, Affinity / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D050556:Fatty Acid-Binding Proteins; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000302:isolation & purification; D036103:Nanotechnology / Q000379:methods; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000032:analysis / Q000302:isolation & purification*; D014170:Transformation, Genetic
D000345:Affinity Labels; D002352:Carrier Proteins; D050556:Fatty Acid-Binding Proteins; D008164:Luminescent Proteins; D011994:Recombinant Proteins; C452700:streptavidin-binding peptide; D049452:Green Fluorescent Proteins; D015500:Chloramphenicol O-Acetyltransferase; D000469:Alkaline Phosphatase