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βII L2079P Mutational Effect on Spectrin (αβ)2 Tetramer Formation Natalie Palmer UIC REU 2010
Acknowledgements • Professor Leslie Fung • Andrea Antoniou, Ph.D. student • Professors Takoudis and Jursich • All REU members • NIH • NSF • DOD • EEC-NSF Grant # 0755115 UIC REU 2010
Spectrin • Cytoskeletal protein that gives stability to cells • Brain spectrin plays a critical role in neuronal growth and secretion • 2 α isoforms • αI, αII • 5 β isoforms • βI, βII, βIII, βIV, βV UIC REU 2010
Organization UIC REU 2010
Project Description • Site directed mutagenesis of bII. Mutation: L2079P • In red blood cell b spectrin (bI), the corresponding residue is proline and the region downstream from the proline residue is unstructured. However, the region in brain bII appears to be helical. We propose that the mutation from Leu to Pro will result in a conformational change in bII and thus function differently. UIC REU 2010
Mutant Primer Design • First Design • A L E R L T T L E • WT: 5’ GCC CTG GAA AGG CTG ACT ACA TTG GAG 3’ • A L E R P T T L E • MT: 5’ GCC CTG GAA AGG CCG ACT ACA TTG GAG 3’ • Second Design • A L E R L T T L E • WT: 5’ GCC CTG GAA AGG CTG ACT ACA TTG GAG 3’ • A L E R P T T L E • MT: 5’ GCC CTG GAA AGG CCT ACT ACA TTG GAG 3’ StuI restriction site UIC REU 2010
Plasmid Preparation and Transformation • Preparation • Polymerase Chain Reaction (PCR) • Denature DNA • Anneal primers to parent strand • Extent primers • DpnI digestion • Removes parent strand • Plasmid analysis • Transformation • Mutant DNA plasmid inserted into DH5α competent cells • Grow cells UIC REU 2010
Plasmid DNA Analysis • DNA gel electrophoresis • Agarose gel • Send to RRC for sequencing • Analyze sequencing results UIC REU 2010
Transformation - BL21 • Same as transformation to DH5α cells used for DNA analysis. • BL21 competent cells are designed for optimal protein expression. • Select colonies for “freeze down” to be used for high-density cell growth. UIC REU 2010
Whole Cell Electrophoresis • Low Molecular Weight standard used to calibrate protein migration on gel. • IPTG added to induce protein expression • SDS-PAGE—buffers used in gel influence the electrophoretic mobility • Proteins migrate on the gel depending on their size • Heavier migrates slower 1 with IPTG 1 w/o IPTG 2 with IPTG 2 w/o IPTG LMWS LMWS kD 97 66 45 30 14.4 UIC REU 2010
Protein Preparation • Suspend cells with our protein in lysis buffer • Sonicate cells to release proteins • Centrifuge to remove pellet • Load supernatant on affinity column to immobilize our protein (fused to GST) to column resin • Elute our fusion protein (GST-bII L2079P) with GSH buffer UIC REU 2010
Purification Results βII L2079P LMWS Lysate Wash 3 4 5 6 7 8 9 out kD 97 66 45 30 98% pure 20.1 14.4 Elution Profile • Obtained 1 g of cells • Collected fusion protein from column in fractions • Plotted concentration vs. fraction number • Ran polyacrylamide gel on fractions with highest concentrations UIC REU 2010
Preparationof αII-N3 • αII-N3 is the binding partner ofβII • Prepared for use in isothermal titration calorimetry (ITC) • Same preparation as βII L2079P LMWS Lysate Wash 2 3 4 5 6 7 out kD 97 66 45 30 14.4 87% pure UIC REU 2010
Preparation of βII WT • Prepared for use in isothermal titration calorimetry (ITC) • Same preparation as βII L2079P and αII Wash out kD LMWS Lysate 2 3 4 5 6 7 8 97 66 45 30 96% pure 14.4 UIC REU 2010
Isothermal Titration Calorimetry (ITC) Schematic http://www.biotechniques.com/multimedia/archive/00037/BTN_A_04376TE01_O_37301a.pdf UIC REU 2010 UIC REU 2010
ITC A + B C Kd = [C] / [A] [B] B is titrated into A, in large volume in isothermal cell. First titration point, all B introduced is associated with A. Heat of association is measured. At the end of the titration, B introduced is not associated with A and no heat released. UIC REU 2010
ITC Continued Our Results • ITC directly measures the binding affinity, binding stoichiometry, and change in enthalpy [GST-αII 1-359] = 35 μM, 1.5 mL [GST-βII L2079P] = 2.1 μM, 7 μL per titration UIC REU 2010 UIC REU 2010
Future Work • Protein Analysis • Do ITC experiments with protein samples and proper controls (WT) • Determine Kd values for βII L2079P with aII in the presence and absence of a inhibitory protein, rG5 or F11, using bII WT with aII as controls, to determine the effect of mutation on βII function UIC REU 2010 UIC REU 2010
What I learned • Research is a trial and error process; it can be frustrating when something goes wrong, but you have to work through the problems and when you get it right it is a very rewarding experience
Questions? UIC REU 2010