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lyh447

无虫 (小有名气)

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注册: 2009-09-05

[交流] DGKzeta基因方面一段+2009年12月23日

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 DGKwas obtained with primers 5’TTTTCATATGGAGCCGCGGGACCCCAG3’ and 5’
TTTTGTCGACTACACAGCTGTCTCCTGGTCC3’. DGKwith all four ankyrin repeats deleted (DGKa) was obtained with primers 5’TTTTGAATTCATGGAGCCGCGGGACCCCAG3’ AND
5’TTTTGTCGACAGTGCGGCATCCCCCTGCAG3’. The ankyrin repeats of DGK(DGKa) 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 32 liters of PBS, pH 7.4, 2 mM dithiothreitol, 10 % glycerol at 4oC
for 36 hours. DGK, DGKa and DGKa was subcloned in frame into pGEX-5X-1 (Amersham
Pharmacia Biotech), expressed in bacterial strain BL21, and purified as GST-DGKa with a glutathione
Sepharose 4B column.

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