Molecular biology procedures

3.2.1.1. Designing and preparation of DNA constructs

- P2X₂ constructs. For yeast two hybrid (Y2H) assays the cDNA sequence corresponding to the full cytoplasmic domain of P2X2 comprising amino acids 355-472 (P2X2CD) was amplified by PCR using full length P2X2 cDNA as template. In addition, the sequence coding for P2X2 splice variant cytoplasmic domain comprising amino acids 355-403 (P2X2(b)CD) was also amplified by PCR using the P2X2(b) cDNA as template. For both constructs the oligonucleotides utilized were N° 2178 and N° 2181 which carried EcoRI and BamHI sites, respectively. The obtained PCR products were cloned in the pLexN vector using the EcoRI/BamHI sites. Employing the P2X2CD in pLexN construct as template, several additional constructs were performed. The sequence coding for amino acid 355-416, 417-472, 355-400, and 401-472 of P2X2CD were amplified by PCR with its corresponding primers, listed on Appendix, and cloned in pLexN vector through EcoRI/BamHI sites.

For recombinant protein expression the sequences coding for P2X2CD and P2X2(b)CD were amplified by PCR with oligonucleotides N° 2184 and N° 2185 containing BamHI and NotI sites respectively, and the digested PCR products were cloned in frame in pGEX-4T-1

vector (Amersham Biosciences) and pET-32a(+) vector (Novagen) which generate the protein fused to Gluthatione-S-transferase (GST) and Thioredoxine (Trx), respectively.

- Fe65 constructs. The sequences coding for Fe65 fragments comprising amino acids 218-479 (Fe65-202) was amplified by PCR using primers N° 2196 and N° 2197 on the full length Fe65 cDNA as template. The PCR product was digested with EcoRI /BamHI and cloned in pVP16-4 vector. From the resultant construct Fe65-202 in pVP16-4 vector, a number of deletions were carried out, thus the sequences coding for amino acids 218-309, 218-284, 255-284 and 285-479 of Fe65 were amplified by PCR with the corresponding primers, listed on Appendix, and cloned in pVP16-4 vector using the same strategy as before.

For recombinant protein expression the sequence coding for amino acids 218-479, 197-255 and 40-100 were amplified by PCR employing the corresponding oligonucleotides, listed on Appendix, and cloned in frame both into the pGEX-4T-1 and pET-32a(+) vectors employing the same strategy as with the P2X2CD.

- PCR reactions. The PCR reactions were performed typically in 50 μl of final volume. The standard PCR mixture contents were as follows: 1X pfu polimerase buffer (Promega),1 μΜ of both sense and antisense primers, 0.8 mM dNTP mixture, 10 ng of the template DNA and 1 unit of the DNA pfu polymerase (Promega). The amplification was started with incubation for 5 minutes at 95^oC followed by 25 amplification cycles of denaturing, annealing, and polymerization with parameters depending on the nature of the primer and on the size of the cDNA to amplify. The amplified fragments were subjected to electrophoresis in agarose gels and the band of interest was purified using the QIAEX II DNA purification kit (Qiagen).

3.2.1.2. Purification, cloning, and isolation of DNA constructs

- Agarose Gel Electrophoresis. The DNA fragments were separated by agarose gel electrophoresis. The 1-2% agarose gels were prepared with 1 X TBE buffer. The gels were stained with ethidium bromide (EtBr; 0,5 μg/ml) and the DNA bands were visualized under ultraviolet light (λ 340nm). The EtBr stained DNA bands were excised from the gel and purified by the QIAEX II kit.

- Preparation of PCR products and vectors for cloning. The purified PCR products and vectors were treated with the corresponding restriction endonuclease to prepare the fragments with cohesive ends, for efficient cloning. Digestion were performed typically in 50 μl final volume containing 1,5-3 μg plasmid DNA or purified PCR product, 1X corresponding buffer (OPA or A-, B-, L-, M-, H- Buffer), and 0.5 U/μl of DNA restriction enzyme. The reactions were performed at 37°C for 2h. After digestion, inactivation of the enzyme was performed by incubation at 65 or 85°C for 15 min, depending on the enzyme employed. The sticky ends of the digested vectors were dephosphorylated by alkaline phosphatase (Promega) during 1h at 37°C, to prevent self-ligation of the vector.

The DNA fragments were separated by agarose gel electrophoresis and purified by the QIAEX II DNA purification kit prior to the ligation.

- Transformation of E.coli Competent Cells. Ligations were performed with a rate of 3:1 (digested PCR product : digested vector), 1X ligation buffer and 1U of T4-DNA ligase (Promega) at 10 μl final volume for 1 h at RT or O.N. at 4°C. The ligation mixture was used directly for transformation of E. coli DH5α or BL21(DE 3) chemically competent cells. 50 μl of competent cells were defrost on ice and mixed with 2 μl of the ligation reaction. After incubation for 30 minutes on ice, the cells were heat-shocked for 1min at 42°C and chilled for 3 min on ice. Following, 500 µl of LB-medium were added and bacteria were incubated for 1 h at 37°C with gentle agitation. Finally, cells were plated onto LB-agar plates containing

appropriate antibiotics (100 μg/ml ampicillin or 50 μg/ml kanamicin) and grown overnight at 37°C.

- DNA isolation.

Small-scale preparation of plasmid DNA (Miniprep). MACHEREY-NAGEL NucleoSpin™ Plasmid Kit was used to purify small amounts of plasmid DNA. For this, 5 ml of LB medium containing the appropriate antibiotics were inoculated with a single E. Coli colony and incubated for 10-16 h at 37°C, 220 rpm. Bacterial cultures were centrifuged (1500 x g, 10 min at 4°C), and the pellet was treated according to the manufacturer’s protocol.

Large-scale preparation of plasmid DNA (Midiprep). For the preparation of large-scale bacterial cultures, 5 ml of LB medium containing appropriate antibiotics were inoculated with a single colony and incubated for 6-8 h at 37°C, 220 rpm. Thereafter, 100 ml of LB-medium with the appropriate antibiotic were inoculated with 0.2 ml of this culture and incubated overnight at 37°C, 220 rpm. The plasmid DNA was isolated with Midipreps DNA purification system Wizard^R Plus (Promega) according to the user’s manual.