Epitope tags recognized by specific binders
Inventors
Frey, Steffen • Goetzke, Hansjoerg • Opazo, Davilla Luis Felipe • Stenmark, Paal Erik Gustav • Martinez, Carranza Markel
Assignees
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Publication Number
US-12365705-B2
Publication Date
2025-07-22
Expiration Date
Abstract
The present invention provides peptides useful as epitope tags, which may be fused to a polypeptide of interest, as well as antibodies that specifically bind to these peptides. The peptides and/or antibodies can be used for detecting, immobilizing, isolating or purifying a molecule that is conjugated to such a peptide and/or antibody.
Core Innovation
The patent describes a peptide-based ALFA epitope-tag system that uses ALFAST peptide (SEQ ID NO: 04) and ALFA PE tag peptide (SEQ ID NO: 33) together with ALFA Selector-ST and ALFA Selector-PE resins. The ALFA tags are small, largely neutral, α-helical peptide tags fused to polypeptides for detection, immobilization, isolation, and purification of tagged polypeptides.
The system further includes camelid single-domain nanobodies that specifically bind the ALFA peptides, including NbALFAST and NbALFAPE, with defined VHH sequences and corresponding CDR1, CDR2, and CDR3 sequences. The disclosed binding relationship enables affinity modulation and native peptide competition for elution.
The document reports performance of the ALFA tag and cognate nanobodies in pull-downs and co-IPs from complex lysates, immunofluorescence detection, intracellular co-localization, and Western and dot-blot sensitivity compared to other tags. It also describes resin behavior under stringent washing and pH challenges, resistance to high salt, chaotropes, detergents, and DTT, and use of ALFA Selector resins for enriching live CD62L+ lymphocytes from PBMCs.
Claims Coverage
The document contains two independent claim families defining 2 inventive features: a single domain antibody that binds the peptide SRLEEELRRRLTE (SEQ ID NO: 04) with specified CDR sequence options, and a single domain antibody comprising one of two specifically defined VHH sequences (SEQ ID NO: 133 or SEQ ID NO: 134).
Single domain antibody binding SRLEEELRRRLTE with selected CDR sequences
A single domain antibody capable of binding a peptide having the sequence SRLEEELRRRLTE (SEQ ID NO: 04), wherein the antibody comprises CDR1, CDR2, and CDR3 sequences selected from specified CDR combinations.
Single domain antibody comprising a specified VHH sequence
A single domain antibody comprising the VHH sequence selected from the group consisting of SEQ ID NO: 133 and SEQ ID NO: 134.
Overall claim coverage centers on single domain antibodies that bind SRLEEELRRRLTE (SEQ ID NO: 04) using specified CDR combinations, and on single domain antibodies defined by specific full VHH sequences (SEQ ID NO: 133 or SEQ ID NO: 134).
Stated Advantages
Strong Western and dot-blot sensitivity versus other tags.
Compatibility with common fixation conditions.
The system retains ALFA-tagged proteins during stringent washes and enables native elution by competition with free ALFAST peptide.
Spontaneous elution is negligible without the competitor peptide.
The ALFA Selector resins and tag recognition show resistance to high salt, chaotropes, detergents, DTT, and multiple pH values.
The system enables specific pull-downs from E. coli and HeLa lysates.
The system is compatible with native co-immunoprecipitation of the YfgM-PpiD complex using ALFA Selector-PE.
Enrichment of live CD62L+ lymphocytes from PBMCs.
Documented Applications
Detection of ALFA-tagged polypeptides, including immunofluorescence detection and intracellular/intrabody co-localization.
Immobilization, isolation, and purification of ALFA-tagged polypeptides.
Pull-downs and co-IPs from complex lysates using ALFA peptide tags and corresponding nanobodies.
Specific pull-downs from E. coli and HeLa lysates.
Native co-immunoprecipitation of the YfgM-PpiD complex using ALFA Selector-PE.
Enriching live CD62L+ lymphocytes from PBMCs by peptide elution and evaluating populations using FACS analysis.
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