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CXCR4 Introduction • G protein coupled receptor (GPCR) • Activated by SDF-1, a chemokine • Thought to be important to two major diseases • HIV- Acts as co-receptor for M Tropic • Possible cure for M Tropic infections • Cancer- Expressed more in breast and other cancers • Aids in metastasis of cancer to lungs and bones •Stop CXCR4 from working, metastasis would stop to lungs and bones Project • The purpose of the project was to mutate the CXCR4 Constitutively Active Mutant (CAM) receptor, do experiments in order to determine whether or not the receptor is active given the random mutations, and to find out exactly where and what the mutations are. • Experiments: 1) Filter Lift Assay 2) His Growth Assay 3) Plasmid Manipulation 4) Restriction Digest 5) Fluorescent Lac Z 6) PCR 7) Western Blot 8) Sequencing 9) Mutagenesis 10) 3-D Modeling Filter Lift Assay • • • • Quickest and easiest method to mass screen for receptor activity Yeast is grown on an agar plate with a grid in order to identify the colonies. The colonies are the lifted with a piece of filter paper that is pre-soaked in X Gal and Z Buffer. The yeast are permeabilised and then incubated over 2-3 hours and the active colonies turned blue. Yeast with an active receptor synthesize B-Galactosidase via FUS-1 activation. X Gal is a substrate for B-Gal. Complete X Gal is colorless; cleaved X Gal is blue. XXX XXXXXX XXXXXXXXX XXXXXX XXX His Growth Assay • Yeast is grown in a medium that lacks a necessary amino acidHistidine. This test is used to confirm the filter lift. If the yeast grows then it is active. His Growth Assay of Single Mutants 0.75 OD 600 • wt n119s M63L1 V82G2 V82G1 V82G3 F248I1 F248I2 0.50 0.25 0.00 0 10 20 Time (Hours) 30 F248I3 Plasmid Manipulation • The plasmid from the revert mutants is extracted by minipreps. • The plasmid is then transformed into Nova Blue competent cells • The DNA is extracted from the competent cells and used for downstream experiments and sequencing. Selection Procedure • Different mediums select different plasmids i.e.: CP4258 –Leu CP1584 –Trp • Different antibiotics select different plasmids in bacteria i.e.: Cp4258 & CP1584 are grown in Ampicillin Restriction Digest • • Cuts plasmid and shows whether or not CXCR4 is present The plasmid is cut with restriction enzymes ( XbaI and NcoI) NcoI- CCATGG XbaI- TCTAGA PCR • • • • A polymerase is added to DNA and replicates the desired DNA. The primers have complementary sequences that line up with the desired segment of the DNA. Taq polymerase adds nucleotides to make up the DNA. Used to make sure CXCR4 is present. Fluorescent Lac Z Same principle as filter lift. FDG is used as substrate in place of X Gal. When cleaved, FDG fluoresces. Fluorescent Lac Z Assay of Single Mutants 75000 Fluorescence • • • 50000 25000 0 wt N119S M63L1 M63L2 M63L3 Mutants V82G1 V82G2 V82G3 F248I1 Western Blot • • • • Shows if protein is present in sample. 9E10 is the antibody that binds to Myc tagged CXCR4. Used to see the size of protein present. Results came out blank. Sequencing • DNA is sent to be sequenced. • Results where mutations, if any, are present. • Mutations that we found: Revert 2-13: V82G Revert 2-16: V82G Revert 2-21: M63L, S312P Revert 2-26: S123I, E288G Revert 2-32: F29S, V59A, F248I, V320E Revert 2-49: F248I Overall Mutation Rate: Mutation Rate: 1/1089 1/1089 2/1089 4/1089 4/1089 2/1089 14/6532= .21% Mutagenesis • • • • Site directed mutagenesis of CXCR4 CAM Separates multiple mutations Mutate into pcDNA3.1zeo(+) for transfection into Mammalian Cells Mutations: M63L V82G F248I E288G 3-D Modeling • Displayed mutations on CXCR4 • Saw possible interactions with other residues • Possible important hydrophobic interactions: S123: W252, N119 L244: L127, S123, Y76, I126 L246: H228, L301 F248: Y256, L253, I215, L127 PLATE 1 PLATE 2 PLATE 3 PLATE 4 PLATE 5 PLATE 6 PLATE 7 PLATE 8 PLATE 9 TOTAL Filter Lift 51 44 47 45 45 48 49 47 48 424 His Growth 0 36 47 38 44 41 0 40 0 246 Yeast Miniprep 0 33 43 21 43 31 0 37 0 208 Bact Transformation 0 6 5 0 0 11 Bact MinipreP 0 6 5 0 0 11 Sal 1 and bact transf. Miniprep 0 6 5 0 0 11 Yeast TRAnsformation (lac Z) 0 6 0 0 6 Fluorescent lac Z 0 6 0 0 6 Sequenced 0 6 0 0 6 Site Direct Mutagenesis (single mutations) 0 4 0 0 4 Yeast Transformation (His, LacZ, FUI) 0 0 0 0 His Growth 0 0 0 0 FUI 0 0 0 0 Fluorescent lac Z 0 0 0 0