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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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