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• W1998173688 abstract "P-glycoprotein (Pgp; mouse MDR3) was expressed inPichia pastoris, grown in fermentor culture, and purified. The final pure product is of high specific ATPase activity and is soluble at low detergent concentration. 120 g of cells yielded 6 mg of pure Pgp; >4 kg of cells were obtained from a single fermentor run. Properties of the pure protein were similar to those of previous preparations, except there was significant ATPase activity in absence of added lipid. Mutant mouse MDR3 P-glycoproteins were purified by the same procedure after growth of cells in flask culture, with similar yields and purity. This procedure should open up new avenues of structural, biophysical, and biochemical studies of Pgp. Equilibrium nucleotide-binding parameters of wild-type mouse MDR3 Pgp were studied using 2′-(3′)-O-(2,4,6-trinitrophenyl)adenosine tri- and diphosphate. Both analogs were found to bind withK d in the low micromolar range, to a single class of site, with no evidence of cooperativity. ATP displacement of the analogs was seen. Similar binding was seen with K429R/K1072R and D551N/D1196N mutant mouse MDR3 Pgp, showing that these Walker A and B mutations had no significant effect on affinity or stoichiometry of nucleotide binding. These residues, known to be critical for catalysis, are concluded to be involved primarily in stabilization of the catalytic transition state in Pgp. P-glycoprotein (Pgp; mouse MDR3) was expressed inPichia pastoris, grown in fermentor culture, and purified. The final pure product is of high specific ATPase activity and is soluble at low detergent concentration. 120 g of cells yielded 6 mg of pure Pgp; >4 kg of cells were obtained from a single fermentor run. Properties of the pure protein were similar to those of previous preparations, except there was significant ATPase activity in absence of added lipid. Mutant mouse MDR3 P-glycoproteins were purified by the same procedure after growth of cells in flask culture, with similar yields and purity. This procedure should open up new avenues of structural, biophysical, and biochemical studies of Pgp. Equilibrium nucleotide-binding parameters of wild-type mouse MDR3 Pgp were studied using 2′-(3′)-O-(2,4,6-trinitrophenyl)adenosine tri- and diphosphate. Both analogs were found to bind withK d in the low micromolar range, to a single class of site, with no evidence of cooperativity. ATP displacement of the analogs was seen. Similar binding was seen with K429R/K1072R and D551N/D1196N mutant mouse MDR3 Pgp, showing that these Walker A and B mutations had no significant effect on affinity or stoichiometry of nucleotide binding. These residues, known to be critical for catalysis, are concluded to be involved primarily in stabilization of the catalytic transition state in Pgp. P-glycoprotein (also known as “multidrug-resistance protein”) transmembrane domain nucleotide-binding site (NBS1 and NBS2 refer to N- and C-terminal sites, respectively) n-dodecyl-β-d-maltoside N-ethylmaleimide 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole 2′-(3′)-O-(2,4,6-trinitrophenyl)adenosine tri- and diphosphate phenylmethanesulfonyl fluoride dithiothreitol nitrilotriacetic acid weight 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid P-glycoprotein (Pgp)1 is a plasma membrane-located, ATP-driven drug efflux pump that confers multidrug resistance on mammalian cells (1Gottesman M.M. Pastan I. Annu. Rev. Biochem. 1993; 62: 385-427Crossref PubMed Scopus (3567) Google Scholar, 2Gros P. Buschman E. Int. Rev. Cytol. 1993; 137: 169-197Crossref Scopus (6) Google Scholar, 3Shapiro A.B. Ling V. J. Bioenerg. Biomembr. 1995; 27: 7-13Crossref PubMed Scopus (61) Google Scholar, 4Sharom F.J. J. Membr. Biol. 1997; 160: 161-175Crossref PubMed Scopus (417) Google Scholar, 5Stein W.D. Physiol. Rev. 1997; 77: 545-590Crossref PubMed Scopus (241) Google Scholar). It occurs commonly in human tumors and is a major obstacle to successful chemotherapy. Consisting of two duplicated “halves” and a total length of around 1280 residues, it is a prominent member of the ABC transporter superfamily (6Higgins C.F. Annu. Rev. Cell Biol. 1992; 8: 67-113Crossref PubMed Scopus (3386) Google Scholar) and shows the typical ABC transporter domain arrangement consisting of two transmembrane domains (TMD) and two nucleotide-binding sites (NBS), in a linear sequence that can be represented as TMD1-NBS1-TMD2-NBS2. Current studies in many laboratories are aimed at understanding Pgp structure, function, normal physiology, and pharmacology. Our laboratory has studied the two nucleotide sites, and we have established that both are catalytic MgATP hydrolysis sites, which interact together closely (7Urbatsch I.L. Sankaran B. Weber J. Senior A.E. J. Biol. Chem. 1995; 270: 19383-19390Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar, 8Urbatsch I.L. Sankaran B. Bhagat S. Senior A.E. J. Biol. Chem. 1995; 270: 26956-26961Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar, 9Sankaran B. Bhagat S. Senior A.E. Biochemistry. 1997; 36: 6847-6853Crossref PubMed Scopus (62) Google Scholar, 10Senior A.E. Bhagat S. Biochemistry. 1998; 37: 831-836Crossref PubMed Scopus (105) Google Scholar). Earlier, we proposed (11Senior A.E. Al-Shawi M.K. Urbatsch I.L. FEBS Lett. 1995; 377: 285-289Crossref PubMed Scopus (429) Google Scholar) a catalytic mechanism for Pgp, in which the two NBS hydrolyze MgATP alternately, and hydrolysis is coupled to drug transport from an inner-facing, higher affinity, drug-binding site to an outer-facing, lower affinity site. Subsequent reports (12Urbatsch I.L. Beaudet L. Carrier I. Gros P. Biochemistry. 1998; 37: 4592-4602Crossref PubMed Scopus (126) Google Scholar, 13Szabo K. Welker E. Bakos Muller M. Roninson I. Varadi A. Sarkadi B. J. Biol. Chem. 1998; 273: 10132-10138Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 14Dey S. Ramachandra M. Pastan I. Gottesman M.M. Ambudkar S.V. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10594-10599Crossref PubMed Scopus (359) Google Scholar, 15Ramachandra M. Ambudkar S.V. Chen D. Hrycyna C.A. Dey S. Gottesman M.M. Pastan I. Biochemistry. 1998; 37: 5010-5019Crossref PubMed Scopus (241) Google Scholar, 16Hyrcyna C.A. Ramachandra M. Ambudkar S.V. Ko Y.H. Pedersen P.L. Pastan I. Gottesman M.M. J. Biol. Chem. 1998; 273: 16631-16634Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar) have supported and extended this proposed mechanism. Further work (17Loo T.W. Clarke D.M. J. Biol. Chem. 1997; 272: 31945-31948Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 18Hafkemeyer P. Dey S. Ambudkar S.V. Hyrcyna C.A. Pastan I. Gottesman M.M. Biochemistry. 1998; 37: 16400-16409Crossref PubMed Scopus (80) Google Scholar, 19Demeule M. Laplante A. Murphy G.F. Wenger R.M. Beliveau R. Biochemistry. 1998; 37: 18110-18118Crossref PubMed Scopus (46) Google Scholar, 20Greenberger L.M. J. Biol. Chem. 1993; 268: 11417-11425Abstract Full Text PDF PubMed Google Scholar) on the structure and location of the drug-binding sites has shown that certain transmembrane α-helices are involved in forming these sites, and work (21Bolhuis H. van Veen H.W. Molenaar D. Poolman B. Driessen A.J.M. Konings W.N. EMBO J. 1996; 15: 4239-4245Crossref PubMed Scopus (178) Google Scholar) with the closely related LmrA protein showed that transport is indeed from the inner lipid leaflet to the outer surface as proposed in Ref. 22Gottesman M.M. Cancer Res. 1993; 53: 747-754PubMed Google Scholar. The mechanism of coupling of energy of ATP hydrolysis to transport of drugs is not well understood, however, at the present time. A preliminary structure of Pgp at low resolution has been published (23Rosenberg M.F. Callaghan R. Ford R.C. Higgins C.F. J. Biol. Chem. 1997; 272: 10685-10694Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar), and, clearly, future structure resolution will be pivotal to more complete understanding of function. Studies of structure and function of proteins depend on availability of pure material. Many of the advances made in the Pgp field so far have come from utilization of the multidrug-resistant Chinese hamster ovary cell model, originally devised by Ling and colleagues (24Bech-Hansen N.T. Till J.E. Ling V. J. Cell. Physiol. 1976; 88: 23-32Crossref PubMed Scopus (239) Google Scholar). By using enriched plasma membranes or purified Pgp (23Rosenberg M.F. Callaghan R. Ford R.C. Higgins C.F. J. Biol. Chem. 1997; 272: 10685-10694Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 25Shapiro A.B. Ling V. J. Biol. Chem. 1994; 269: 3745-3754Abstract Full Text PDF PubMed Google Scholar, 26Liu R. Sharom F.J. Biochemistry. 1997; 36: 2836-2843Crossref PubMed Scopus (83) Google Scholar, 27Urbatsch I.L. Al-Shawi M.K. Senior A.E. Biochemistry. 1994; 33: 7069-7076Crossref PubMed Scopus (226) Google Scholar) from such cells, an extensive body of literature has been built up, dealing with enzymatic, transport, and structural properties of Pgp and using a range of biochemical and biophysical approaches. The Chinese hamster ovary cell model has two drawbacks, however, which are that it is too expensive to scale up to truly large scale production of pure Pgp, and mutant proteins cannot be purified. Mutagenesis has been successfully employed in studies of Pgp in membrane preparations from insect (Sf9) or cultured mammalian cells and has proved extremely valuable, for example see Refs. 13Szabo K. Welker E. Bakos Muller M. Roninson I. Varadi A. Sarkadi B. J. Biol. Chem. 1998; 273: 10132-10138Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar and28Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 22957-22961Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 29Azzaria M. Schurr E. Gros P. Mol. Cell. Biol. 1989; 9: 5289-5297Crossref PubMed Scopus (270) Google Scholar, 30Hrycyna C.A. Airan L.E. Germann U.A. Ambudkar S.V. Pastan I. Gottesman M.M. Biochemistry. 1998; 37: 13660-13673Crossref PubMed Scopus (94) Google Scholar. However, there is no method so far for purifying mutant Pgp in significant amounts. Earlier methods (31Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 21449-21452Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar) produced only 10-μg amounts of protein, and more recent methods, although improved (12Urbatsch I.L. Beaudet L. Carrier I. Gros P. Biochemistry. 1998; 37: 4592-4602Crossref PubMed Scopus (126) Google Scholar), have not yet provided large quantities (>200 μg). Among promising methods for further scale-up, expression of Pgp in yeast cells appears foremost. At time of writing, Pgp has been purified from Saccharomyces cerevisiae (32Mao Q. Scarborough G.A. Biochim. Biophys. Acta. 1997; 1327: 107-118Crossref PubMed Scopus (26) Google Scholar) and Pichia pastoris (12Urbatsch I.L. Beaudet L. Carrier I. Gros P. Biochemistry. 1998; 37: 4592-4602Crossref PubMed Scopus (126) Google Scholar, 33Beaudet L. Urbatsch I.L. Gros P. Methods Enzymol. 1998; 292: 397-413Crossref PubMed Scopus (35) Google Scholar), with the latter system shown to be amenable to expression and purification of mutant Pgp (12Urbatsch I.L. Beaudet L. Carrier I. Gros P. Biochemistry. 1998; 37: 4592-4602Crossref PubMed Scopus (126) Google Scholar). In this paper we have scaled up purification of Pgp using growth ofP. pastoris cells in a fermentor and a new purification method. The final material is pure, active in detergent-soluble form, and activated to high specific ATPase activity on addition of minimal amounts of lipid. Wild-type mouse MDR3 Pgp and several mutant mouse MDR3 proteins containing substitutions in the Walker A and Walker B consensus sequences were purified by the same procedure. The purified Pgp is readily amenable to fluorescence analysis. We report the nucleotide-binding properties of wild-type and mutant Pgp using the fluorescent nucleotides TNP-ATP and TNP-ADP, and we demonstrate that mutations in the Walker A and B sequences that fully impair catalysis have little or no effect on equilibrium nucleotide binding. n-Dodecyl-β-d-maltoside with low intrinsic fluorescence was from Anatrace (catalog number D310). Imidazole with low intrinsic fluorescence was from Sigma (catalog number I-0250). Ni2+-NTA agarose resin was from Qiagen. C219 monoclonal antibody was from Signet Laboratories.Escherichia coli lipids were from Avanti (acetone/ether-precipitated, catalog number 100600). Asolectin (crude soybean phospholipid) was from Sigma. TNP-ATP and TNP-ADP (sodium salts) were obtained from Molecular Probes. Antifoaming agent MAZU DF204 (catalog number 558958) was from BASF Corp. All other chemicals were from recognized sources. A strain carrying wild-type mouse MDR3 cDNA incorporated into the chromosome behind the AOX1 promoter was described previously (12Urbatsch I.L. Beaudet L. Carrier I. Gros P. Biochemistry. 1998; 37: 4592-4602Crossref PubMed Scopus (126) Google Scholar, 33Beaudet L. Urbatsch I.L. Gros P. Methods Enzymol. 1998; 292: 397-413Crossref PubMed Scopus (35) Google Scholar). Strains carrying the following mutations were described (12Urbatsch I.L. Beaudet L. Carrier I. Gros P. Biochemistry. 1998; 37: 4592-4602Crossref PubMed Scopus (126) Google Scholar): K429R, K1072R, and K429R/K1072R (Walker A single and double mutants); D551N, D1196N, and D551N/D1196N (Walker B single and double mutants). In all cases, the Pgp contained a C-terminal six His-tag to facilitate purification. The P. pastoris cell line GS115 (Invitrogen, license number 145 457) was used and grown according to the supplier's guidelines. Media were as follows: MGY, 1.34% w/v yeast nitrogen base without amino acid (Difco), 1% v/v glycerol, 0.4 mg/liter biotin; MM, same as MGY but with 0.5% v/v methanol replacing glycerol. Conditions for growth of P. pastoris GS115 cells transformed with wild-type or mutant mouse MDR3 Pgp were as described previously (33Beaudet L. Urbatsch I.L. Gros P. Methods Enzymol. 1998; 292: 397-413Crossref PubMed Scopus (35) Google Scholar,34Beaudet L. Urbatsch I.L. Gros P. Biochemistry. 1998; 37: 9073-9082Crossref PubMed Scopus (45) Google Scholar). Briefly, a single colony from an MGY agar plate was inoculated into 10 ml of liquid MGY medium and incubated at 30 °C with shaking to A 600 of 2.0 (∼18 h), then this 10 ml was inoculated into 1 liter of MGY medium in a 2-liter baffled Fernbach flask. Incubation at 30 °C with shaking toA 600 of 2.0 was continued (∼24 h), then the cells were centrifuged in sterile bottles (2500 × gfor 10 min at 23 °C). The cells were resuspended toA 600 of 2.0 in 0.5% v/v methanol-containing MM medium, and 1 liter was added to a 2-liter baffled Fernbach flask covered with cheesecloth. Incubation continued for 72 h with additions of methanol (0.5% v/v) at 24 and 48 h. Cells were finally collected by centrifugation at 2500 × g for 10 min at 23 °C and resuspended in ice-cold “homogenization buffer” (0.33 m sucrose, 300 mm Tris-Cl, pH 7.4, 1 mm EDTA, 1 mm EGTA, 2 mm DTT, 100 mm 6-aminohexanoic acid) at a concentration of 0.5 g wet wt cells/ml buffer. The fermentation was done according to Invitrogen guidelines in a BiofloIV bench-top fermenter (New Brunswick Scientific) equipped with a 20-liter water-jacketed stainless steel vessel and microprocessor control of pH, dissolved oxygen, agitation, temperature, and nutrient-feed and with electronic foam control. The temperature was kept at 29 °C. The vessel containing medium composed of 0.93 g/liter CaSO4, 18.2 g/liter K2SO4, 14.9 g/liter MgSO4·7H2O, and 4% v/v glycerol in 10-liter volume was sterilized for 30 min at 122 °C, then 9 g/liter (NH4)2SO4, 25 g/liter sodium hexametaphosphate, and 4.25 ml/liter trace salts (Invitrogen, PTM1) were added aseptically. Fermentation proceeded in three phases. To begin the initial “glycerol batch phase,” 1-liter of start-up culture was added. The start-up culture consisted of 1 liter of cells grown in flasks in MGY, as described above. Agitation was maintained at a minimum of 500 rpm and was programmed to increase up to 1000 rpm to maintain a dissolved oxygen above 40% of the initial setting (100%). A pH of 5.0 was maintained using 14% (w/v) ammonium hydroxide. The glycerol batch phase was run until glycerol was completely consumed (24 h), i.e. whenA 600 was stable for 2 h. A yield of 104 g wet wt cells/liter was achieved at this stage. The second phase was the “glycerol-fed phase,” that was initiated by continuously feeding the culture with a medium containing 50% (v/v) glycerol and 12 ml/liter PTM1 trace salts. The feed rate was 18.15 ml/h/liter and was carried out until cell density was 213 g wet wt cells/liter (4–5 h). The feed was stopped for 2 h to ensure total glycerol depletion. The third phase was the “methanol-fed phase.” The methanol feed, containing 12 ml of PTM1 trace salts per liter of methanol, was initiated at the rate of 1 ml/h/liter of initial fermentation volume for 13 h, was increased to 2 ml/h/liter for 24 h, then further to 2.4 ml/h/liter for 24 h, and finally to 3 ml/h/liter for the rest of the methanol-fed phase. This phase lasted in total for 6 days. A sample of cells was taken daily and checked for A 600, cell density (wet wt cells/liter), and Pgp expression. The cells reached a cell density of 425 g/liter on day 6. The fermenter was cooled to 10 °C prior to harvesting. The cells were centrifuged at 2500 × g for 10 min at 4 °C, washed twice with distilled water, and frozen at −70 °C in 15% (v/v) glycerol in water. A rapid procedure was used to obtain a crude membrane fraction from cells grown in the fermentor or flasks, to monitor Pgp expression. Cells from 10 ml of flask culture or 0.5–1 ml of fermentor culture were centrifuged at 2000 ×g for 5 min and resuspended in 0.5 ml of homogenization buffer (above). The suspension was transferred to a 2-ml microcentrifuge tube and centrifuged at 2000 × g for 5 min. The pellet was resuspended in 300 μl of homogenization buffer containing 10 μg/ml leupeptin and pepstatin A, 2.5 μg/ml chymostatin, and 1 mm PMSF, and then 100 μl of acid-washed glass beads (Sigma G-9268, 425–600 μm) were added. The mixture was vortexed vigorously six times for 1 min each, with 2 min on ice between each vortexing. The supernatant was spun at 2000 ×g for 2 min to remove beads, then at 14,000 ×g for 20 min, and then finally the remaining supernatant was spun at 250,000 × g for 60 min. The pellet (“rapid membranes,” 100–200 μg of protein) was dissolved in 2% (w/v) SDS, analyzed by protein assay, SDS-gel electophoresis, and Western blotting with C219 antibody against Pgp. For flask cultures, frozen cells in homogenization buffer were rapidly thawed at 30 °C. For fermentor cultures, cells were thawed, centrifuged at 2500 × g for 10 min, and then resuspended in ice-cold homogenization buffer at a concentration of 0.5 g wet wt cells per ml. All further steps were at 4 °C or on ice. Protease inhibitors were added as follows: 2 μg/ml pepstatin A and leupeptin, 0.5 μg/ml chymostatin, 1 mm PMSF. Cells were passed one to three times through a French press set at 20,000 pounds/square inch, adding fresh PMSF (1 mm) every 25 min and adjusting the pH to 7.4 after each pass with 1 m Tris-Cl, pH 7.4. The homogenate was centrifuged at 3,500 × g for 15 min, and the pellet was discarded, and the supernatant was centrifuged at 14,000 × g for 30 min. These steps removed unbroken cells, nuclei, and mitochondrial fractions, which contained little Pgp as shown by Western blotting. The supernatant was centrifuged at 200,000 × g for 90 min, and the pellet containing the microsomes was resuspended in buffer (50 mm Tris-Cl, pH 7.4, 10% v/v glycerol containing 2 μg/ml pepstatin A and leupeptin, 0.5 μg/ml chymostatin, 1 mm PMSF), to the same volume as before the 200,000 × g centrifugation. Resuspension was achieved initially by use of a paintbrush and then by passage through a 22-gauge needle, followed by 10 strokes by hand in a Potter-Elvehjem Teflon pestle homogenizer. The microsomes were recentrifuged at 200,000 × g for 60 min and resuspended as above, this time in “buffer A” consisting of 50 mm Tris-Cl, pH 8.0, 50 mm NaCl, 30% v/v glycerol, 1 mm 2-mercaptoethanol, 10 mmimidazole, plus protease inhibitors as follows: 2 μg/ml pepstatin A and leupeptin, 0.5 μg/ml chymostatin, 1 mm PMSF. At this stage the microsomes could be stored frozen at −70 °C. Yield of microsomes was 40–50 mg per 10 g wet wt cells. To 1 volume of thawed microsomes (4 mg protein/ml) was added an equal volume of 1.2% w/vn-dodecyl-β-d-maltoside (DM) in buffer A plus protease inhibitors as above. The suspension was mixed by inversion and allowed to sit on ice for 10 min. It was then centrifuged at 60,000 × g for 30 min. Ni2+-NTA agarose resin (Qiagen) was pre-equilibrated in buffer A and added to the supernatant from the DM solubilization at a ratio of 1 ml of packed resin per 100 mg of solubilized microsomal protein. The slurry was incubated at 4 °C for 16–20 h on a Labquack rotator. The resin with bound Pgp was transferred into a column and washed with 25-bed volumes of buffer A containing 0.1% (w/v) DM and then with 5 volumes of buffer A containing 0.1% DM plus 20 mm imidazole. Pgp was eluted with 3 volumes of buffer A containing 0.1% DM plus 200 mm imidazole. The 200 mmimidazole eluate from the Ni2+-NTA column was diluted 4-fold with buffer B (10 mm Tris-Cl, pH 8.0, 30% v/v glycerol, 1 mm 2-mercaptoethanol, 0.1% DM) and applied to a column of Whatman DE52 cellulose, which had been precycled according to the manufacturer's instructions and equilibrated in buffer B. The column volume was 2 ml of resin per 100 mg of solubilized microsomal protein. Pgp did not adsorb and was collected in the flow-through. The column was washed with 2 column volumes of buffer B, and the wash fraction was pooled with the flow-through. This material contained pure Pgp. Elution of the DE52 column with 300 mm NaCl in buffer B showed that all the contaminant proteins in the eluate from the Ni2+-NTA column step were adsorbed to the DE52 column. Pgp was concentrated by ultrafiltration under N2 using an Amicon concentrator with a YM10 or XM50 membrane membrane to a final concentration of 1–2 mg/ml. The concentrated material was aliquoted and stored at −70 °C. Lipid stocks were made from asolectin or E. coli lipids by adding 40 mm Tris-Cl, pH 7.4, 0.1 mm EGTA, to solid lipid and sonicating in a bath sonicator at 4 °C until the suspension clarified (final concentration was 20 mg of lipid/ml). This stock was stored under N2. For activation, Pgp and lipid were mixed at the appropriate ratio (w/w) in 40 mm Tris-Cl, pH 7.4, 0.1 mm EGTA, with DTT (2 mm), and incubated 20 min at 23 °C, then sonicated in a bath sonicator at 4 °C for 30 s. The protein was stored on ice for the minimum period until assay. Fluorescence measurements were made in a SPEX Fluorolog 2 fluorimeter. For TNP-nucleotide binding experiments, using stirred 2-ml cuvettes at room temperature, excitation wavelength was 408 nm, and emission spectra from 500 to 600 nm were collected. Pgp was activated as above using 2/1 ratio of asolectin/Pgp (w/w) before addition to the cuvette. Final Pgp concentration in the cuvette was 140 nm. Control cuvettes containing every component except Pgp (including lipid and detergent) were used to correct for inner filter and volume effects. The buffer was 50 mm Tris-SO4, pH 7.5, with 2 mm MgCl2 (“with Mg2+”) or with 0.5 mm EDTA (“without Mg2+”). TNP-nucleotides were added, and the fluorescence spectra were taken when equilibration was reached (≤3 min). Concentrations of stock solutions of TNP-nucleotides were determined by measurement of absorption at 408 nm using an extinction coefficient of 26,400m−1 cm−1 (35Hiratsuka T. Uchida K. Biochim. Biophys. Acta. 1973; 320: 635-647Crossref PubMed Scopus (143) Google Scholar). Titration curves were fit using nonlinear regression to a model assuming a single class of sites, which in all cases gave a satisfactory fit to the data. Further details of individual experiments are given in figure legends. SDS-gel electrophoresis on 8 or 10% gels used the Bio-Rad Minigel system with manufacturer supplied gels. Western blotting followed standard procedures and used the monoclonal anti-Pgp C219 antibody (Signet Laboratories). ATPase assay at 37 °C by continuous linked-enzyme procedure was as in Ref. 7Urbatsch I.L. Sankaran B. Weber J. Senior A.E. J. Biol. Chem. 1995; 270: 19383-19390Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar. Protein estimation was by the BCA (36Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Goekie N.M. Olson B.J. Klenk D.C Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (18713) Google Scholar) or Bradford (37Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217548) Google Scholar) methods. The procedure is described in detail under “Experimental Procedures.” A strain of P. pastoris expressing wild-type mouse MDR3 protein (12Urbatsch I.L. Beaudet L. Carrier I. Gros P. Biochemistry. 1998; 37: 4592-4602Crossref PubMed Scopus (126) Google Scholar, 33Beaudet L. Urbatsch I.L. Gros P. Methods Enzymol. 1998; 292: 397-413Crossref PubMed Scopus (35) Google Scholar) was grown in the fermentor. After initiation of the methanol feed to induce Pgp expression, small samples were taken at daily intervals for analysis of Pgp. Cells were broken, and a rapid membrane preparation was made as described under “Experimental Procedures.” SDS-gel electrophoresis and Western blotting were performed. Pgp expression was evident first at 24 h after beginning the methanol feed, and the Pgp level remained constant as a fraction of total membrane protein each 24 h for 6 days (data not shown). However, cell density increased steadily from 234 g/liter at day 1 to 425 g/liter at day 6, thus yielding a total of over 4 kg of cells at harvesting on day 6 after methanol induction. Previously Gros and colleagues (12Urbatsch I.L. Beaudet L. Carrier I. Gros P. Biochemistry. 1998; 37: 4592-4602Crossref PubMed Scopus (126) Google Scholar) purified Pgp from the same P. pastoris strain, from cells grown in flask culture, using a procedure involving preparation of purified plasma membranes by sucrose gradient centrifugation, solubilization of Pgp from purified plasma membranes, Ni2+-NTA (His-tag) chromatography, and reconstitution into proteoliposomes. Here we adapted this procedure as follows. First, we avoided purifying plasma membranes, because we were dealing with large amounts of cells, and also we wished to harvest any Pgp resident in intracellular membrane locations. Thus we prepared a microsome fraction and solubilized Pgp from it directly. Second, we changed the solubilization conditions, omitting addition of exogenous lipid. Third, we added a DEAE-cellulose chromatography step, which removed all contaminants in material obtained at the Ni2+-NTA step. Fourth, we omitted reconstitution in proteoliposomes, so that the final product would be detergent-soluble and monodisperse for fluorescence studies. Table I (columns 2 and 3) shows a typical purification of wild-type mouse MDR3 Pgp from a 120-g batch of fermentor-grown cells, which yielded 5.8 mg of final product. The procedure has been repeated many times with excellent reproducibility. Yield of pure Pgp ranged from 1% of microsomal protein (as in Table I) up to 1.5% and was usually close to 0.05 mg of Pgp per g wet wt cells. Fig. 1 shows a Coomassie Blue-stained SDS gel documenting the purification procedure, with the final pure Pgp shown in lane 7. We have run larger amounts of pure Pgp (up to 10 μg) on the gel and have seen no contaminants. The very minor band just visible at around 60 kDa on the gel is a proteolysis product from Pgp, as demonstrated by Western blotting with C219 antibody and also vanadate trapping with 8-azido-[α-32P]ATP. At the stage of detergent solubilization, the average distribution of total protein was around 80% in the supernatant and 20% in the pellet. Western blotting showed that the supernatant was enriched in Pgp so that extraction of Pgp was efficient. Recovery of Pgp at the Ni2+-NTA and DE52 chromatography steps is excellent. The band in the 300 mm NaCl eluate from the DE52 column which runs at the same position as Pgp (Fig. 1, lane 8) was shown to be Pgp by Western blotting. Thus a small amount of Pgp adsorbs to the DE52 column, but the bulk clearly did not adsorb under our conditions. The final pure Pgp after concentration to 1–2 mg/ml is in a buffer containing 0.1% DM and behaves as a soluble protein. It is stable to freezing at −70 °C; however, loss of activity was noted on multiple freezing and thawing.Table IPurification of wild-type and mutant mouse mdr3 PgpWild-typeMutant1-aPurification of mutant K429R/K1072R is described. Other mutants as listed in Fig. 2 legend showed the same behavior. ATPase activity was very low to zero in all the mutants.YieldATPase1-bATPase was assayed in presence of 150 μmverapamil and after activation with DTT and lipid (E. colilipid or asolectin at 2/1 w/w ratio).Yieldmgunits/mgmgCells120,00040,000Microsomes564 (100)167 (100)DM-soluble supernatant487 (86)140 (84)Eluate from Ni2+-NTA column with 200 mmimidazole10.6 (1.9)3.24.1 (2.5)DE52 column flow-through after concentration5.8 (1.0)4.91.6 (1.0)1-a Purification of mutant K429R/K1072R is described. Other mutants as listed in Fig. 2 legend showed the same behavior. ATPase activity was very low to zero in all the mutants.1-b ATPase was assayed in presence of 150 μmverapamil and after activation with DTT and lipid (E. colilipid or asolectin at 2/1 w/w ratio). Open table in a new tab Typically the pure Pgp was divided into aliquots and stored at −70 °C. Data described are for material frozen and thawed once. Preincubation with DTT was necessary to achieve maximal activity, commonly an enhancement of 5- to 8-fold was seen, and this" @default.
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• W1998173688 title "Large Scale Purification of Detergent-soluble P-glycoprotein fromPichia pastoris Cells and Characterization of Nucleotide Binding Properties of Wild-type, Walker A, and Walker B Mutant Proteins" @default.
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