1. 1 Human metabotropic glutamate receptor 6: Expression and purification Kalyan Tirupula Graduate Student JKS Lab, UPitt

2. 2 pMT3 vector carrying metabotropic glutamate receptor 6 (hmGluR6 or mGluR6 or GRM6) Construct is a gift from Dr. Phyllis R. Robinson, University of Maryland, Baltimore County. [GRM6 cloned into pMT3 by Ben Nickel, Grad student, Dr. Phyllis Robinson Lab]

3. 3 pMT3-hMGlur6: 1 » 7796 R5: 996 « 1608 (complementary) GLU6FOR: 1035 » 1717 NM_000843: 1067 » 3700 R4: 1424 « 2021 (complementary) R3: 1870 « 2441 (complementary) R2: 2249 « 2866 (complementary) R1: 2672 « 3302 (complementary) GLU6REV: 3096 « 3798 (complementary) 150010001500200025003000350040004500500055006000650070007500 1 2 2 1 Nucleotide 2196 (A  G); No effect on translation (Thr732) Insertion of 1D4 tag just before stop codon Sequence verification of mGlur6 clone Sequencing results confirm that the clone is accurate !

4. 4 Transfection and Solubilization experiments DEAE-Dextran based transfection method was adopted. COS1 cells are used for transfection Solubilization experiments were set up in : –CHAPS (1%), DM (1%), Triton (1%) and OG (4%) Supernatants Cell pellets Cells were harvested 84hrs after transfection. Solubilization experiments inconclusive as it was done Over Night (> 12 hrs), which is usually recommended because of probable protein degradation or aggregation.

5. 5 Solubilization experiments continued … Solubilization done for ~1 hr @ 4C in CHAPS (1%), DM (1%), Triton (1%) and OG (4%) SupernatantsCell pellets Solubilization in 4% OG is comparatively better. Boiling samples before running on the gel induces aggregation.

6. 6 Time Course transfection Experiments for determining optimal time for cell harvest Supernatants Cell pellets 24 48 55 72 96 Cells were harvested at 24, 48, 55, 72 and 96 hrs after transfection mGLur6 expression 48-55 hrs post- transfection seems optimal For all the future experiments unless specified, the cells were harvested 55 hrs after transfection.

7. 7 Affinity purification of mGluR6 using 1D4 sepharose beads – pH and Buffer optimization

8. 8 mGluR6 purification experiment continued …. mGluR6 elutes with 50mM Tris + 150mM NaCl + 0.88% OG + 70uM 9mer + pH 8.0

9. 9 pH and Buffer optimization continued …. mGluR6 elution with 50mM NaHCO3 + 0.88% OG + 70uM 9mer @ pH 8.4 seems to elute high molecular weight aggregates

10. 10 pH and Buffer optimization continued.. 250 150 100 75 KDa mGluR6 elution with 50mM Tris + 0.88% OG + 70uM 9mer @ pH 8.0 seems to be optimal

11. 11 Future Directions mGlur6 reconstitution into ‘native’ lipid environment mGluR6 activity assays –Need to confirm that purified mGluR6 retains activity Immediate attention to make a stable cell line Large scale GRM6 purification for future experiments –Structural dynamics (NMR) on binding of native and non native allosteric modulators

12. 12 Acknomledgements I sincerely thank….. –Judith for the useful discussions and guidance to perform the experiments. I also thank –David for all the feedback for protein purification experiments. –Harpreet Dhiman for guidance with cell culture –Hussein for helping with some gels recently –All the JKS lab members for a friendly and successful work environment.

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