![]() of unique characteristics of the underlying FN3 scaffold: small (90 residues). It is small (76 amino acids, 8.5kDa), stable, and highly. Lane 1, protein ladder Lane 2 & 5, pre-enrichment mixture Lane 3 & 6, enrichment using SH2-1.8 Lane 4 & 7, enrichment using SH2-3.1. Monobodies are simple and robust alternative to antibodies in creating. Ubiquitin is a natural protein of human origin (non-immunogenic). Antibody-like fragments are attractive as protein scaffolds for developing novel therapeutics due to their smaller size (12-50 kDa) compared with full-sized. Treated denotes 16 hrs of phosphatase treatment prior to enrichment. describe the mechanisms underlying differences between how two engineered influenza hemagglutinin immunogens elicit broadly cross-reactive antibodies targeting a conserved epitope. Non-treated denotes no phosphatase treatment prior to enrichment. Lane 1, molecular weight marker Lane 2 & 6, pre-enrichment mixture Lane 3 & 7, enrichment with SH2-1.8 Lane 4 & 8, enrichment with SH2-3.1 Lane 5 & 9, enrichment without SH2 mutants (D) Selective enrichment from 1:1 BSA-P:C4-sTyr-GST mixture. Lanes 6 to 9, enrichment of non-sulfated protein, C4-Tyr-GST, in the presence of 100-fold BSA using engineered SH2 mutants. Lanes 2 to 5, enrichment of sulfoprotein, C4-sTyr-GST, in the presence of 100-fold BSA using engineered SH2 mutants. ![]() Serial dilutions of C4-sTyr-GST were detected by 1 μg/mL of SH2-1.8, SH2-3.1, or 1/1000 dilution of anti-sTyr monoclonal antibody (C) Enrichment experiments. 1 μg of designated protein detected using various concentrations of SH2-1.8 or SH2-3.1 (B) Dot blot analysis of limit of detection. (A) Dot blot analysis of effective concentrations and substrate specificity of SH2 domain mutants. Utilities of the evolved SH2 mutants were demonstrated by the detection and enrichment of sulfoproteins. An alternative approach is to design molecular scaffolding systemsi.e. Further molecular docking simulations highlight potential mechanisms supporting observed characteristics of these SH2 mutants. Using tailored selection schemes, several SH2 mutants are identified with high affinity and specificity to sulfotyrosine. ![]() In this work, we seek to engineer the phosphotyrosine binding pocket of a Src Homology 2 (SH2) domain to act as an antisulfotyrosine antibody mimic. Enrichment of sulfotyrosine-containing proteins (sulfoproteins) from complex biological samples are typically required before analysis. Because of its unstable nature under mass spectrometry conditions and residing on low-abundance cell surface proteins, sulfated tyrosine (sulfotyrosine) residues are difficult to detect or analyze. Protein tyrosine O-sulfation is an essential post-translational modification required for effective biological processes such as hemostasis, inflammatory response, and visual phototransduction. ![]()
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