Expression and purification of membrane proteins Initial 膜蛋白的表达和初步纯化.pptVIP

Expression and purification of membrane proteins: Initial screening of Thermotoga maritima α-helical membrane proteins for NMR structural studies Expression Assay Detergent Screen Soluble ≠ folded! Summary Acknowledgements * The membrane proteome of T. maritima 24% 76% Membrane proteins Cytosolic proteins Percentage of Membrane Proteins in T. maritima Electron micrograph of T. maritima. The arrow points to the outer membrane, the “toga”, for which the organism is named. 55% 20% 25% Proteins with unknown function Other functionally annotated proteins ABC transporters Functional Annotations of the Membrane Proteome Target selection Determine localization Insoluble fraction Membrane fraction Optimize expression to the membrane Refold Extract Co2+ or Ni2+-affinity purification Detergent stability screen NMR spectroscopy Assay expression in E.coli Overall Approach to Preparing Membrane Protein Samples for NMR Studies # of proteins 0 200 400 600 800 1000 1200 1400 1600 Protein length (amino acids) Target Selection Size distribution of the membrane proteome NMR sample requirements 600 mL of 1 mM protein uniform 15N and 13C-labeling monodisperse MW 30 kD (for standard NMR experiments, development of TROSY has extended the MW limit for NMR studies) Selected fifty targets less than 16 KD (~130 aa) that contained one to four predicted transmembrane segments. 21.5 14.4 6.0 kD Eleven out of fifty targets overexpressed using arabinose induction. Para 6-aa Pml I 6-his CAC GTG TM gene TTA ATT AA Pac I Pme I Nco I RBS Sma I PT7 Expression Vector 96 x 65 mL Fermentor Localization of target membrane proteins In order to verify that the target proteins are membrane proteins, the E. coli membranes were isolated using ultracentrifugation and extracted with n-decyl-?-D-maltoside . Of the eleven expressing targets, two expressed exclusively in the insoluble fraction (IB) and nine expressed to both the