Human metabotropic glutamate receptor 6: Expression and purification

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

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. GLU6REV: 3096 « 3798 (complementary) R1: 2672 « 3302 (complementary) R2: 2249 « 2866 (complementary) R3: 1870 « 2441 (complementary) R4: 1424 « 2021 (complementary) GLU6FOR: 1035 » 1717 Nucleotide 2196 (A  G); No effect on translation (Thr732) 1 R5: 996 « 1608 (complementary) 2 1 NM_000843: 1067 » 3700 Insertion of 1D4 tag just before stop codon 2 1 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 6000 6500 7000 7500 pMT3-hMGlur6: 1 » 7796 Sequence verification of mGlur6 clone Sequencing results confirm that the clone is accurate !

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

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

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

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

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. 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. 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. 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.