ABCMdb

PMID: 21424391

Jacobs A, Emmert D, Wieschrath S, Hrycyna CA, Wiese M
Recombinant synthesis of human ABCG2 expressed in the yeast Saccharomyces cerevisiae: an experimental methodological study.
Protein J. 2011 Mar;30(3):201-11., [PubMed]

Sentences

No. Mutations Sentence Comment

4 ABCG2 p.Arg482Gly In this work we describe a recombinant synthesis of human ABCG2 R482G from S. cerevisiae. Login to comment 5 ABCG2 p.Arg482Gly We expressed the human ABCG2 R482G variant in S. cerevisiae and purified the protein from total yeast membranes. Login to comment 22 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly Mutation of arginine-482 to threonine or glycine considerably extends the spectrum of transported substrates. Login to comment 23 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly The variants R482T or R482G transport additional substrates such as rhodamine 123 and doxorubicin, whereas the recognition of other substrates such as Hoechst 33342 and pheophorbide A remains unaffected [10, 17]. Login to comment 24 ABCG2 p.Arg482Gly One of these variants (R482G) was chosen for purification experiments described in this manuscript showing the above mentioned broader substrate recognition. Login to comment 38 ABCG2 p.Arg482Gly We have tried to apply the same system to human ABCG2 (R482G variant). Login to comment 41 ABCG2 p.Arg482Gly We were able to show function by stimulation of ATPase activity with prazosin and other known substrates of ABCG2 (R482G) such as sulfasalazine and progesterone [15, 19]. Login to comment 44 ABCG2 p.Arg482Gly 2 Materials and Methods 2.1 Expression Vector pCHH10m3N-ABCG2 Human ABCG2 cDNA (R482G variant) was cloned into the pCHH10m3N plasmid [2], where the expression was under control of the constitutive phosphoglycerate kinase (PGK) promoter. Login to comment 100 ABCG2 p.Arg482Gly ABCG2 p.Arg482Gly 3 Results and Discussion 3.1 Expression of ABCG2 (R482G) in S. cerevisiae The ABCG2 variant R482G was chosen, as it displays altered substrate specificity in comparison to wild-type (wt) ABCG2. Login to comment 143 ABCG2 p.Arg482Gly Functionality of the purified ABCG2 (R482G) was analyzed by a drug-stimulated ATPase assay using the standard stimulator prazosin. Login to comment 166 ABCG2 p.Arg482Gly Following solubilization trials, purification of ABCG2 from transformed LPY11-ABCG2 (R482G) cells was performed with 2% FC-16 as described for OG. Login to comment 234 ABCG2 p.Arg482Gly Position in ABCG2 R482G Sequence 675.42 379-383 R.WVSKR.S 801.48 158-163 K.NERINR.V 820.48 178-184 K.VGTQFIR.G 863.48 359-365 K.KKITVFK.E 900.52 466-473 R.VSSYFLGK.L 970.58 324-331 K.QDKPLIEK.L 987.62 315-323 K.ATEIIEPSK.Q 1004.60 418-426 K.NDSTGIQNR.A 1014.62 164-172 R.VIQELGLDK.V 1020.58 48-56 K.LKSGFLPCR.K 1037.54 379-386 R.WVSKRSFK.L 1044.62 87-96 K.SSLLDVLAAR.K 1145.70 148-157 R.LATTMTNHEK.N 1148.64 138-147 R.ENLQFSAALR.L 1172.66 87-97 K.SSLLDVLAARK.D 1231.68 62-72 K.EILSNINGIMK.P 1292.78 252-263 K.LFDSLTLLASGR.L 1296.66 347-358 K.AELHQLSGGEKK.K 1358.74 315-326 K.ATEIIEPSKQDK.P 1360.68 50-61 K.SGFLPCRKPVEK.E 1397.74 161-172 R.INRVIQELGLDK.V 1399.76 617-628 K.QGIDLSPWGLWK.N 1424.68 347-359 K.AELHQLSGGEKKK.K 1433.70 332-343 K.LAEIYVNSSFYK.E 1462.78 178-191 K.VGTQFIRGVSGGER.K 1514.86 164-177 R.VIQELGLDKVADSK.V 1560.68 454-465 K.LFIHEYISGYYR.V 1601.88 48-61 K.LKSGFLPCRKPVEK.E 1791.82 332-346 K.LAEIYVNSSFYKETK.A 1962.91 173-191 K.VADSKVGTQFIRGVSGGER.K 2141.11 347-365 K.AELHQLSGGEKKKKITVFK.E 2247.19 173-193 K.VADSKVGTQFIRGVSGGERKR.T 2253.16 97-118 R.KDPSGLSGDVLINGAPRPANFK.C 2301.22 360-378 K.KITVFKEISYTTSFCHQLR.W 2464.36 62-86 K.EILSNINGIMKPGLNAILGPTGGGK.S 2537.14 231-251 R.MSKQGRTIIFSIHQPRYSIFK.L 2603.23 366-386 K.EISYTTSFCHQLRWVSKRSFK.L 2923.36 148-172 R.LATTMTNHEKNERINRVIQELGLDK.V 2941.39 332-357 K.LAEIYVNSSFYKETKAELHQLSGGEK.K 2957.35 360-383 K.KITVFKEISYTTSFCHQLRWVSKR.S 3194.56 387-417 K.NLLGNPQASIAQIIVTVVLGLVIGAIYFGLK.N 4862.00 315-357 K.ATEIIEPSKQDKPLIEKLAEIYVNSSFYKETKAELHQLSGGEK.K Masses were matched employing a tryptic in-silico digest using the GPMAW 8.0 software (Lighthouse Data) standard compound prazosin, and other known stimulators of ATPase activity, such as progesterone and sulfasalazine, increased the liberation of inorganic phosphate from ATP. Login to comment