ABCMdb

PMID: 19285030

Wan L, Liang X, Huang Y
Characterization of the ATPase activity of a novel chimeric fusion protein consisting of the two nucleotide binding domains of MRP1.
Arch Biochem Biophys. 2009 May 15;485(2):102-8. Epub 2009 Mar 11., 2009-05-15 [PubMed]

Sentences

No. Mutations Sentence Comment

4 ABCC1 p.Glu1455Gln Additionally, the E1455Q mutant was found to be nonfunctional. Login to comment 33 ABCC1 p.Glu1455Gln In order to introduce the E1455Q mutation into NBD2 of the pET21a-NBD1-GST-NBD2 construct, primers 50 -GAA GAT CCT TGT GTT GGA TCA GGC CAC GGC AGC CGT GGA CCT GG-30 and 50 -CCA GGT CCA CGG CTG CCG TGG CCT GAT CCA ACA CAA GGA TCT TC-3` were used, and pET21a-NBD1-GST-NBD2 construct was used as template in the mutagenesis using QuikChange site directed mutagenesis kit (Stratagene). Login to comment 114 ABCC1 p.Glu1455Gln To this end, we constructed a NBD1-GST-NBD1 fusion protein and an E1455Q mutant of the NBD1-GST-NBD2 fusion protein. Login to comment 116 ABCC1 p.Glu1455Gln Similar to isolated NBD1 and NBD2, neither NBD1-GST-NBD1 nor NBD1-GST-NBD2/E1455Q showed any detectable ATPase activity (Fig. 4B). Login to comment 138 ABCC1 p.Glu1455Gln (B) ATPase activities of the chimeric fusion protein NBD1-GST-NBD2, the NBD1(+)NBD2 mixture, the NBD1(+)GST-NBD2 mixture, NBD1-GST-NBD1, and NBD1-GST-NBD2/E1455Q, all purified from Superdex 200 columns. Login to comment 163 ABCC1 p.Asp1454Leu ABCC1 p.Lys684Leu ABCC1 p.Glu1455Leu ABCC1 p.Lys1333Leu ABCC1 p.Glu1455Gln Among the NBD1-GST-NBD2 mutants, K684L in Walker A of NBD1, K1333L in Walker A of NBD2, and D1454L/E1455L in Walker B of NBD2 were expressed mainly as inclusion bodies in E. coli, and only the E1455Q mutant was expressed in a sufficient quantity of soluble protein to allow activity analysis. Login to comment 164 ABCC1 p.Glu1455Gln The results clearly showed that the NBD1-GST-NBD2/ E1455Q mutant was not functional, consistent with a previous report on this null-mutant in the intact MRP1 [22]. Login to comment