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Human metabotropic glutamate receptor 6:

Expression and purification

Kalyan Tirupula

Graduate Student

JKS Lab, UPitt

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pMT3+mGlur67796 bp

hmGluR6

1D4

STOP

Eco RI (1062)

Mlu I (3726)

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]

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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)

1 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 6000 6500 7000 7500

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Fragment of pMT3-hMGlur6 new3451 bp (molecule 7796 bp)

hmGluR6

1D4

GLU6FOR GLU6REVR1R2R3R4R5

STOP

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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 !

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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.

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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.

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Time Course transfection Experiments for determining optimal time for cell harvest

Supernatants Cell pellets

24 48 55 72 96 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.

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

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mGluR6 purification experiment continued ….

mGluR6 elutes with 50mM Tris + 150mM NaCl + 0.88% OG + 70uM 9mer + pH 8.0

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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

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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

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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

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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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