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Amino Acid-Specific Binder And Selectively Identifying An Amino Acid
Abstract
N-terminal amino acid binding (NAAB) reagents are a tool for parallel, high-throughput proteomics. Afluorescently-labeled NAAB allows immobilized peptides to be identified by their N-terminal residues using single-molecule fluorescent microscopy, which enables novel proteomic analysis, like fluorescence-based next-generationprotein sequence. We have engineered a leucine-targeting NAAB by making multiple mutations in a variant of theN-End Rule adaptor protein ClpS protein from the cyanobacterium Thermosynechococcus elongatus. Our collectionof mutations was discovered by screening a yeast-display library of T. elongatus ClpS2 by fluorescence-assisted cellsorting for variants that displayed higher affinity and specificity for the leucine target.
patent description
The invention is recombinant proteins (see attached sequences) which binds to leucine at the N-terminus of peptides/polypeptides/proteins with high affinity and specificity. Compared to the wild-type T. elongatus ClpS2, which served as the starting point for the discovery of the novel sequence, the invention has a significant difference in the binding discrimination for N-terminal leucine vs other N-terminal amino acids (e.g., phenylalanine). The detection of binding activity using these so called NAAB variants indicates an N-terminal leucine with high confidence.