ABSTRACT
[Structure: see text] The synthesis and characterization of thermoresponsive, water-soluble poly-N-isopropyl acrylamide (PNIPAM) derived macroligands displaying cyclosporin A (CsA) and dexamethasone (Dex) for use as novel affinity resins are described. Characterization of these soluble macroligands, including ligand loading and integrity, was determined by 1H NMR spectroscopy. One of the CsA macroligands was used in a protein affinity experiment to capture known binding proteins of CsA, the cyclophilins, from Jurkat T-cell lysates.
Subject(s)
Chromatography, Affinity/methods , Cyclosporine/chemistry , Dexamethasone/chemistry , Ligands , Cyclophilins , Glucocorticoids/chemistry , Humans , Immunosuppressive Agents/chemistry , Jurkat Cells , Molecular Structure , Protein BindingABSTRACT
Uroporphyrinogen III synthase from the cyanobacterium Anacystis nidulans was overproduced in Escherichia coli and analyzed by site specific mutagenesis. Of the nine conserved amino acids altered, only a single tyrosine mutant (Y166F) showed any significant decrease in activity suggesting this residue is critical for proper substrate binding and/or catalysis.
