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DGKzeta基因方面一段+2009年12月23日
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The cDNA fragment of rat Ob-Rbc, obtained by RT-PCR with primers 5’TCACACCAGAGAATGAAAAAG3’ and 5’CACAGTTAAGTCACACATCTTA3’, was used to screen a rat brain two-hybrid library (Clontech). By RT-PCR, the cDNA fragment of rat DGKwas obtained with primers 5’TTTTCATATGGAGCCGCGGGACCCCAG3’ and 5’ TTTTGTCGACTACACAGCTGTCTCCTGGTCC3’. DGKwith all four ankyrin repeats deleted (DGKa) was obtained with primers 5’TTTTGAATTCATGGAGCCGCGGGACCCCAG3’ AND 5’TTTTGTCGACAGTGCGGCATCCCCCTGCAG3’. The ankyrin repeats of DGK(DGKa) was obtained with primers 5’ TTTTGAATTCGCACTGCCCCAAGGTGAAG3’ and 5’TTTTGTCGACTACACAGCTGTCTCCTGGTCC3’. Protein expression and purification. The cDNA fragment of rat Ob-Rac was obtained by RT-PCR with primers 5’TCACACCAGAGAATGAAAAAG3’ and 5’AAGAGTGTCCGCTCTCTTTTG3’. The cDNA fragments for Ob-Rac and Ob-Rbc were subcloned into plasmid pET-32a(+) (Novagen) for expression as thioredoxin (Trx)-fusion proteins in bacterial strain BL21(DE3)pLysS (Novagen). To generate Ob-Rbt, pET-32a(+)-Ob-Rbc was digested by Kpn I, followed by T4 DNA polymerase and ligation. The bacteria expressing Trx-Ob-Rbc and Trx-Ob-Rbt were solubilized in buffer TUNN (10 mM Tris, 8 M urea, 100 mM NaH2PO4, 0.5 % NP-40) plus 5 mM imidazole, pH 7.9. The proteins were purified with a Ni-NTA Superflow column (Qiagen) by washing sequentially with TUNN plus 20 mM imidazole, pH 7.9, and TUNN plus 20 mM imidazole, pH 6.3. The proteins were eluted with TUNN plus 20 mM imidazole, pH 5.6, and renatured by dialysis against 32 liters of PBS, pH 7.4, 2 mM dithiothreitol, 10 % glycerol at 4oC for 36 hours. DGK, DGKa and DGKa was subcloned in frame into pGEX-5X-1 (Amersham Pharmacia Biotech), expressed in bacterial strain BL21, and purified as GST-DGKa with a glutathione Sepharose 4B column. |
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