Is there any system with small molecule binders and a short protein tag that is higher affinity than 6xHIS-NTA?
V5 and Flag tag are the only ones I've seen other than His.
Einhauer A, Jungbauer A. The FLAG peptide, a versatile fusion tag for the purification of recombinant proteins. J Biochem Biophys Methods. 2001 Oct 30;49(1-3):455-65. doi: 10.1016/s0165-022x(01)00213-5. PMID: 11694294.
Also consider the MBP that is engineered with a linker with a protease cut site. MBP sticks to the affinity column, and you use the protease to elute your protein of interest. Then of course the protease has to be purified out, so it should have an affinity tag or biotin.
Wikipedia has a long list.
Peptide tags
- ALFA-tag, a de novo designed helical peptide tag (SRLEEELRRRLTE) for biochemical and microscopy applications. The tag is recognized by a repertoire of single-domain antibodies [5]
- AviTag, a peptide allowing biotinylation by the enzyme BirA and so the protein can be isolated by streptavidin (GLNDIFEAQKIEWHE)
- C-tag, a peptide that binds to a single-domain camelid antibody developed through phage display (EPEA)[6][7]
- Calmodulin-tag, a peptide bound by the protein calmodulin (KRRWKKNFIAVSAANRFKKISSSGAL)
- iCapTag™ (intein Capture Tag), peptide-based a self-removing tag controlled by pH change (MIKIATRKYLGKQNVYGIGVERDHNFALKNGFIAHN). Its patented component derived from Nostoc punctiforme (Npu) intein. This tag is used for protein purification of recombinant proteins and its fragments. It can be used in research labs and it is intended for large-scale purification during downstream manufacturing process as well. The iCapTag™-target protein complex can be expressed in a wide range of expression hosts (e.g. CHO and E.coli cells). It is not intended for fully expressed mAbs or membrane proteins[8][9][10]
- polyglutamate tag, a peptide binding efficiently to anion-exchange resin such as Mono-Q (EEEEEE) [11]
- polyarginine tag, a peptide binding efficiently to cation-exchange resin (from 5 to 9 consecutive R)
- E-tag, a peptide recognized by an antibody (GAPVPYPDPLEPR)
- FLAG-tag, a peptide recognized by an antibody (DYKDDDDK)[12]
- HA-tag, a peptide from hemagglutinin recognized by an antibody (YPYDVPDYA)[13]
- His-tag, 5-10 histidines bound by a nickel or cobalt chelate (HHHHHH)Gly-His-tags are N-terminal His-Tag variants (e.g. GHHHH, or GHHHHHH, or GSSHHHHHH) that still bind to immobilised metal cations but can also be activated via azidogluconoylation to enable click-chemistry applications[14]
- Myc-tag, a peptide derived from c-myc recognized by an antibody (EQKLISEEDL)
- NE-tag, an 18-amino-acid synthetic peptide (TKENPRSNQEESYDDNES) recognized by a monoclonal IgG1 antibody, which is useful in a wide spectrum of applications including Western blotting, ELISA, flow cytometry, immunocytochemistry, immunoprecipitation, and affinity purification of recombinant proteins [15]
- Rho1D4-tag, refers to the last 9 amino acids of the intracellular C-terminus of bovine rhodopsin (TETSQVAPA). It is a very specific tag that can be used for purification of membrane proteins.
- S-tag, a peptide derived from Ribonuclease A (KETAAAKFERQHMDS)
- SBP-tag, a peptide which binds to streptavidin (MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP)[16][17][self-published source?]
- Softag 1, for mammalian expression (SLAELLNAGLGGS)
- Softag 3, for prokaryotic expression (TQDPSRVG)
- Spot-tag, a peptide recognized by a nanobody (PDRVRAVSHWSS) for immunoprecipitation, affinity purification, immunofluorescence and super resolution microscopy
- Strep-tag, a peptide which binds to streptavidin or the modified streptavidin called streptactin (Strep-tag II: WSHPQFEK)[2]
- T7-tag, an epitope tag derived from the T7 major capsid protein of the T7 gene (MASMTGGQQMG). Used in different immunoassays as well as affinity purification Mainly used [18]
- TC tag, a tetracysteine tag that is recognized by FlAsH and ReAsH biarsenical compounds (CCPGCC)
- Ty tag (EVHTNQDPLD)
- V5 tag, a peptide recognized by an antibody (GKPIPNPLLGLDST)[19]
- VSV-tag, a peptide recognized by an antibody (YTDIEMNRLGK)
- Xpress tag (DLYDDDDK), a peptide recognized by an antibody
Hi Michael Ronzetti. Jennifer Washburn provided a very complete answer, from which you can take a lot of conclusions.
I would just like to add that in terms of affinity (dissociation constant), the strongest tags are SBP (low picomolar range), FLAG, HA, MYC, PA (low nanomolar range), and GST (low micromolar range). For comparison, 6xHis-tag is in the high micromolar to millimolar range. Depending on your final goal, hopefully you can make a rational decision based on the information we provided.
Good luck!
Best,
Tomás
The Strep-tag system for one-step purification and high-affinity detection or capturing of proteins | Nature Protocols
Strep-tag® - leading affinity tag in recombinant protein production (iba-lifesciences.com)
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