Abstract
The widespread success of affinity tags throughout the biological sciences has prompted interest in developing new and convenient labeling strategies. Affinity tags are well-established tools for recombinant protein immobilization and purification. More recently these tags have been utilized for selective biological targeting towards multiplexed protein detection in numerous imaging applications as well as for drug-delivery. Recently, we discovered a phage-display selected cyclic peptide motif that was shown to bind selectively to NeutrAvidin and avidin but not to the structurally similar streptavidin. Here, we have exploited this selectivity to develop an affinity tag based on the evolved DRATPY moiety that is orthogonal to known Strep-tag technologies. As proof of principle, the divalent AviD-tag (Avidin-Di-tag) was expressed as a Green Fluorescent Protein variant conjugate and exhibited superior immobilization and elution characteristics to the first generation Strep-tag and a monovalent DRATPY GFP-fusion protein analogue. Additionally, we demonstrate the potential for a peptide based orthogonal labeling strategy involving our divalent AviD-tag in concert with existing streptavidin-based affinity reagents. We believe the AviD-tag and its unique recognition properties will provide researchers with a useful new affinity reagent and tool for a variety of applications in the biological and chemical sciences.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 54-61 |
| Number of pages | 8 |
| Journal | Protein Expression and Purification |
| Volume | 56 |
| Issue number | 1 |
| DOIs | |
| State | Published - Nov 2007 |
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Keywords
- Affinity
- AviD-tag
- Avidin
- His-tag
- Immobilization
- NeutrAvidin streptavidin
- Orthogonal
- Purification
- Strep-tag
ASJC Scopus subject areas
- Biochemistry
Cite this
The AviD-tag, a NeutrAvidin/avidin specific peptide affinity tag for the immobilization and purification of recombinant proteins. / Gaj, Thomas; Meyer, Scott C.; Ghosh, Indraneel.
In: Protein Expression and Purification, Vol. 56, No. 1, 11.2007, p. 54-61.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - The AviD-tag, a NeutrAvidin/avidin specific peptide affinity tag for the immobilization and purification of recombinant proteins
AU - Gaj, Thomas
AU - Meyer, Scott C.
AU - Ghosh, Indraneel
PY - 2007/11
Y1 - 2007/11
N2 - The widespread success of affinity tags throughout the biological sciences has prompted interest in developing new and convenient labeling strategies. Affinity tags are well-established tools for recombinant protein immobilization and purification. More recently these tags have been utilized for selective biological targeting towards multiplexed protein detection in numerous imaging applications as well as for drug-delivery. Recently, we discovered a phage-display selected cyclic peptide motif that was shown to bind selectively to NeutrAvidin and avidin but not to the structurally similar streptavidin. Here, we have exploited this selectivity to develop an affinity tag based on the evolved DRATPY moiety that is orthogonal to known Strep-tag technologies. As proof of principle, the divalent AviD-tag (Avidin-Di-tag) was expressed as a Green Fluorescent Protein variant conjugate and exhibited superior immobilization and elution characteristics to the first generation Strep-tag and a monovalent DRATPY GFP-fusion protein analogue. Additionally, we demonstrate the potential for a peptide based orthogonal labeling strategy involving our divalent AviD-tag in concert with existing streptavidin-based affinity reagents. We believe the AviD-tag and its unique recognition properties will provide researchers with a useful new affinity reagent and tool for a variety of applications in the biological and chemical sciences.
AB - The widespread success of affinity tags throughout the biological sciences has prompted interest in developing new and convenient labeling strategies. Affinity tags are well-established tools for recombinant protein immobilization and purification. More recently these tags have been utilized for selective biological targeting towards multiplexed protein detection in numerous imaging applications as well as for drug-delivery. Recently, we discovered a phage-display selected cyclic peptide motif that was shown to bind selectively to NeutrAvidin and avidin but not to the structurally similar streptavidin. Here, we have exploited this selectivity to develop an affinity tag based on the evolved DRATPY moiety that is orthogonal to known Strep-tag technologies. As proof of principle, the divalent AviD-tag (Avidin-Di-tag) was expressed as a Green Fluorescent Protein variant conjugate and exhibited superior immobilization and elution characteristics to the first generation Strep-tag and a monovalent DRATPY GFP-fusion protein analogue. Additionally, we demonstrate the potential for a peptide based orthogonal labeling strategy involving our divalent AviD-tag in concert with existing streptavidin-based affinity reagents. We believe the AviD-tag and its unique recognition properties will provide researchers with a useful new affinity reagent and tool for a variety of applications in the biological and chemical sciences.
KW - Affinity
KW - AviD-tag
KW - Avidin
KW - His-tag
KW - Immobilization
KW - NeutrAvidin streptavidin
KW - Orthogonal
KW - Purification
KW - Strep-tag
UR - http://www.scopus.com/inward/record.url?scp=34848866248&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=34848866248&partnerID=8YFLogxK
U2 - 10.1016/j.pep.2007.06.010
DO - 10.1016/j.pep.2007.06.010
M3 - Article
VL - 56
SP - 54
EP - 61
JO - Protein Expression and Purification
JF - Protein Expression and Purification
SN - 1046-5928
IS - 1
ER -