Alexander Yaroshchuk

1. Expression of Metabotropic Glutamate Receptors Alexander Yaroshchuk

2. Scheme of mGluR1 and mGluR6 Figure 1. Domain structure of mGluRs investigated here.

3. His-Tag His-Tag Strep-Tag Hind III Constructs used in the work Nde I 516 877 Strep-Tag Nde I Hind III 585 877 Figure 2. mGluR6 constructs in pBlunt Figure 3. Map of pCR-Blunt vector

4. Cloning of the H585 and H516 constructs in pMT4 (for COS-1 cells) and pACMV-tetO (for HEK293) • The H585 and H516 constructs were cut from pBlunt vector by • EcoRI and blunted by Klenow Fragment of DNA Polymerase. • The blunted constructs were cloned into blunted pMT4 and • pACMV-tetO vectors • The clones were checked for the orientation of inserts and • sequenced. • 4. The following plasmids were obtained: 1) pMT4+H585 2) pMT4+H516 3) pACMV-tetO+H585 4) pACMV-tetO+H516

5. Expression checking of H516 construct in COS-1 cells 1 2 3 4 5 6 7 8 9 10 1 - His Marker 2 - Cells Lysate 3 - Supernatant, DM 4 - Pellet, DM 5 - Supernatant, OG 6 - Pellet, OG 7 - Supernatant, CHAPS 8 - Pellet, CHAPS 9 - Supernatant, Sarcosyl 10 - Marker II Figure 4. Western Blot on HIS-tag. Expression of H516 consruct in COS-1 cells was not observed

6. Stable Transfection of HEK293 with H516 and H585 constructs • Cells are grown at the following Geneticin concentrations: • 300 µg/ml and 1000 µg/ml • There are approximately 3 colonies per plate

7. Procedure of mGluR1 repair (1) (2) (3) Cysteinene-rich region Extracellular region Transmembrane region C-terminus region 1D4-Tag N-terminus C-terminus 522 592 841 1199 1 BamHI EcoNI Mutations:(1) 367 Trp to Arg (2) 511 Lys to Glu (3) 725 Ile to Val 1) PCR 1: Start Primer + Primer Repairing Mutation (1) 2) PCR 2: Product of PCR 1 + Primer Repairing Mutation (2) 3) PCR 3: Product of PCR 2 + Primer Containing EcoNI (~a.a. 629) 4) Cloning of the PCR3 fragment into pBlunt vector 5) Sequencing of the clone

8. Procedure of mGluR1 repair(continued) (1) (2) (3) Cysteinene-rich region Extracellular region Transmembrane region C-terminus region 1D4-Tag N-terminus C-terminus 522 592 841 1199 1 BamHI EcoNI Strep-Tag pBlunt+PCR3 EcoNI/MluI fragment R522 EcoNI/MluI + 522 592 841 1199 1 6) Restriction of pBlunt+PCR3 and pBlunt+R522 with EcoNI/MluI 7) Ligation of EcoNI/MluI fragment from pBlunt+R522 with EcoNI/ClaI fragment from pBlunt+PCR3 8) Sequencing of the results (pBlunt+mGluR1strep)

9. Cloning of mGluR1 in pMT4 and pACMV-tetO vector • The mGluR1 construct was cut from pBlunt vector by • BamHI and blunted by Klenow Fragment of DNA Polymerase. • The blunted fragment was cloned into blunted pMT4 and • pACMV-tetO vectors • The clones were sequenced.

10. Conclusion • H585 and H516 constructs were cloned into pMT4 and • pACMV-tetO vectors • Expression of H516 construct was checked in COS-1 cells • no expression was observed. • Stable transfection of HEK293 with H585 and H516 constructs • was done. The cells are still growing. • Rat mGluR1 construct was repaired.

11. Future work • Checking of HEK293 stably transfected with H516 and H585. • Stable transfection of HEK293 with the full-length mGluR1 • Transient transfection of COS-1 cells with the full-length mGluR1 • and H585 construct