Generation of recombinant affinity reagents against a two-phosphosite epitope of ATF2

McGinnis, Jennifer E.; Kay, Brian K.

doi:10.1016/j.nbt.2017.10.004

Cited by 2 publications

(5 citation statements)

References 32 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Correlated with this observation, clones E12 and H11 closely resemble the consensus sequence, and are the strongest binders, whereas clones E1 and B3 are more diverged and are the weakest binders. Among the residues shared most often among the binders is tyrosine at position 83, consistent with previous conclusions that position 83 is extremely important for FHA domain interactions with their phosphopeptide targets [ 23 , 26 , 27 ].…”

Section: Resultssupporting

confidence: 90%

“…Published results demonstrate that the three residues C-terminally adjacent to the pT residue, most notably the +3 residue, contribute significantly to FHA domain interactions with peptides [ 20 , 21 , 23 , 27 ]. While many of the 20 amino acids occur among phosphopeptide ligands for FHA domains, the Akt1 pT308 FHA variant is the first one observed to have glycine at the +3 position.…”

Section: Resultsmentioning

confidence: 99%

“…The phagemid DNA isolated from affinity selection against the Akt1-pT308 peptide were subcloned into the pKP600ΔIII vector [ 30 ] and expressed and purified as described elsewhere [ 23 ]. Protein purity was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and protein concentrations were measured with a NanoDrop A280 spectrophotometer.…”

Section: Methodsmentioning

confidence: 99%

“…Biotinylated peptides diluted in PBS were incubated overnight in 0.5 mM DTT at 4 °C. ELISAs were performed as previously described [ 21 , 23 ] using the peptide targets incubated with dithiothreitol (DTT) at a concentration of 2.5 μM in 100 μL and FHA variants at concentrations varying from 0.01 to 10 μM in PBST. The absorbance was read at 405 nanometers (nm) at 10-min intervals, for a total of 40 min.…”

Section: Methodsmentioning

confidence: 99%

“…Unlike other scaffolds and most antibodies, FHA domains are selective for phosphothreonine (pT)-containing targets due to a pocket on the domain that interacts with phosphate and γ-methyl group of phosphothreonine (pT) [ 21 , 22 ]. Because of this unique characteristic, a phage library displaying FHA1 variants randomized at residues 82–84 in the β4-β5 loop and residues 133–139 in the β10-β11 loop have been employed to generate affinity reagents to a variety of targets [ 20 , 21 , 23 ]. Here, we describe the isolation and characterization of Akt1 phosphothreonine 308 (pT308)-binding reagents.…”

Section: Introductionmentioning

confidence: 99%

See 4 more Smart Citations

A Recombinant Affinity Reagent Specific for a Phosphoepitope of Akt1

McGinnis

Venegas

Lopez

et al. 2018

IJMS

View full text Add to dashboard Cite

The serine/threonine-protein kinase, Akt1, plays an important part in mammalian cell growth, proliferation, migration and angiogenesis, and becomes activated through phosphorylation. To monitor phosphorylation of threonine 308 in Akt1, we developed a recombinant phosphothreonine-binding domain (pTBD) that is highly selective for the Akt1 phosphopeptide. A phage-display library of variants of the Forkhead-associated 1 (FHA1) domain of yeast Rad53p was screened by affinity selection to the phosphopeptide, 301-KDGATMKpTFCGTPEY-315, and yielded 12 binding clones. The strongest binders have equilibrium dissociation constants of 160–180 nanomolar and are phosphothreonine-specific in binding. The specificity of one Akt1-pTBD was compared to commercially available polyclonal antibodies (pAbs) generated against the same phosphopeptide. The Akt1-pTBD was either equal to or better than three pAbs in detecting the Akt1 pT308 phosphopeptide in ELISAs.

show abstract

Section: Resultssupporting

confidence: 90%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

A Recombinant Affinity Reagent Specific for a Phosphoepitope of Akt1

McGinnis

Venegas

Lopez

et al. 2018

IJMS

View full text Add to dashboard Cite

show abstract

Engineered FHA domains can bind to a variety of Phosphothreonine-containing peptides

Thota,

Allen,

Grahn

et al. 2024

Protein Engineering, Design and Selection

View full text Add to dashboard Cite

Antibodies play a crucial role in monitoring post-translational modifications, like phosphorylation, which regulates protein activity and location; however, commercial polyclonal and monoclonal antibodies have limitations in renewability and engineering compared to recombinant affinity reagents. A scaffold based on the Forkhead-associated domain (FHA) has potential as a selective affinity reagent for this post-translational modification. Engineered FHA domains, termed phosphothreonine-binding domains (pTBDs), with limited cross-reactivity were isolated from an M13 bacteriophage display library by affinity selection with phosphopeptides corresponding to human mTOR, Chk2, 53BP1, and Akt1 proteins. To determine the specificity of the representative pTBDs, we focused on binders to the pT543 phosphopeptide (536-IDEDGENpTQIEDTEP-551) of the DNA repair protein 53BP1. ELISA and western blot experiments have demonstrated the pTBDs are specific to phosphothreonine, demonstrating the potential utility of pTBDs for monitoring the phosphorylation of specific threonine residues in clinically relevant human proteins.

show abstract

Generation of recombinant affinity reagents against a two-phosphosite epitope of ATF2

Cited by 2 publications

References 32 publications

A Recombinant Affinity Reagent Specific for a Phosphoepitope of Akt1

A Recombinant Affinity Reagent Specific for a Phosphoepitope of Akt1

Engineered FHA domains can bind to a variety of Phosphothreonine-containing peptides

Contact Info

Product

Resources

About