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High-Quality Protein Crystal Growth of Mouse Lipocalin-Type Prostaglandin D Synthase in Microgravity.

Inaka K, Takahashi S, Aritake K, Tsurumura T, Furubayashi N, Yan B, Hirota E, Sano S, Sato M, Kobayashi T, Yoshimura Y, Tanaka H, Urade Y - Cryst Growth Des (2011)

Bottom Line: Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) catalyzes the isomerization of PGH(2) to PGD(2) and is involved in the regulation of pain and of nonrapid eye movement sleep and the differentiation of male genital organs and adipocytes, etc.Mouse L-PGDS with a C65A mutation was previously crystallized with citrate or malonate as a precipitant, and the X-ray crystallographic structure was determined at 2.0 Å resolution.The precipitant solution was 40% (w/v) PEG 8000, 100 mM sodium chloride, and 100 mM HEPES-NaOH (pH 7.0).

View Article: PubMed Central - PubMed

Affiliation: Maruwa Foods and Biosciences Inc. , 170-1 Tsutsui-cho, Yamatokoriyama, Nara 639-1123, Japan.

ABSTRACT
Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) catalyzes the isomerization of PGH(2) to PGD(2) and is involved in the regulation of pain and of nonrapid eye movement sleep and the differentiation of male genital organs and adipocytes, etc. L-PGDS is secreted into various body fluids and binds various lipophilic compounds with high affinities, acting also as an extracellular transporter. Mouse L-PGDS with a C65A mutation was previously crystallized with citrate or malonate as a precipitant, and the X-ray crystallographic structure was determined at 2.0 Å resolution. To obtain high-quality crystals, we tried, unsuccessfully, to crystallize the C65A mutant in microgravity under the same conditions used in the previous study. After further purifying the protein and changing the precipitant to polyethylene glycol (PEG) 8000, high-quality crystals were grown in microgravity. The precipitant solution was 40% (w/v) PEG 8000, 100 mM sodium chloride, and 100 mM HEPES-NaOH (pH 7.0). Crystals grew on board the International Space Station for 11 weeks in 2007, yielding single crystals of the wild-type L-PGDS and the C65A mutant, both of which diffracted at around 1.0 Å resolution. The crystal quality was markedly improved through the use of a high-viscosity precipitant solution in microgravity, in combination with the use of a highly purified protein.

No MeSH data available.


Related in: MedlinePlus

(a) SDS-PAGE of wild-type mouse L-PGDS (lane 1) and the C65A mutant (lane 2) under reducing conditions. The molecular weight marker proteins are indicated in the right lane. (b) Native-PAGE of wild-type mouse L-PGDS (lane 1) and the C65A mutant (lane 2) under nonreducing conditions. Bands of marker protein (pI = 4.4) are shown on both sides.
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fig1: (a) SDS-PAGE of wild-type mouse L-PGDS (lane 1) and the C65A mutant (lane 2) under reducing conditions. The molecular weight marker proteins are indicated in the right lane. (b) Native-PAGE of wild-type mouse L-PGDS (lane 1) and the C65A mutant (lane 2) under nonreducing conditions. Bands of marker protein (pI = 4.4) are shown on both sides.

Mentions: The cDNAs of wild-type mouse L-PGDS without its N-terminal signal peptide of 24 amino acids and its C65A mutant were expressed in Escherichia coli BL21(DE3) (Invitrogen, CA, USA) as a glutathione (GSH) transferase-fusion protein. The fusion protein was purified by GSH-Sepharose 4B (GE Healthcare, Buckinghamshire, U.K.) column chromatography from a soluble fraction of E. coli cells. After incubation with thrombin (Sigma-Aldrich, WI, USA), L-PGDS protein was obtained and further purified by column chromatography on Superdex 75 (GE Healthcare) in 5 mM Tris-HCl pH 8.0, followed by Mono-S HR5/5 (GE Healthcare) chromatography with a linear gradient from 0 to 500 mM sodium chloride in 20 mM Tris-HCl, pH 7.5, at 293 K. Structural homogeneities of final purified preparations of the wild-type and C65A mutant of mouse L-PGDS were evaluated by SDS-PAGE (under reducing conditions) and by native PAGE (PAGE without a denaturing reagent, such as SDS, and under nonreducing conditions) as shown in Figure 1. The wild-type L-PGDS showed the same PGDS activity as that of the C89,189A mutant.(14) They were concentrated to 40 mg mL–1 in 100 mM HEPES-NaOH, pH 7.0, and 100 mM sodium chloride using a Centricon YM-10 membrane (10,000 nominal molecular weight limit; Millipore, MA, USA) and stored at 277 K. The protein concentration was determined spectrophotometrically at 280 nm.


High-Quality Protein Crystal Growth of Mouse Lipocalin-Type Prostaglandin D Synthase in Microgravity.

Inaka K, Takahashi S, Aritake K, Tsurumura T, Furubayashi N, Yan B, Hirota E, Sano S, Sato M, Kobayashi T, Yoshimura Y, Tanaka H, Urade Y - Cryst Growth Des (2011)

(a) SDS-PAGE of wild-type mouse L-PGDS (lane 1) and the C65A mutant (lane 2) under reducing conditions. The molecular weight marker proteins are indicated in the right lane. (b) Native-PAGE of wild-type mouse L-PGDS (lane 1) and the C65A mutant (lane 2) under nonreducing conditions. Bands of marker protein (pI = 4.4) are shown on both sides.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3105485&req=5

fig1: (a) SDS-PAGE of wild-type mouse L-PGDS (lane 1) and the C65A mutant (lane 2) under reducing conditions. The molecular weight marker proteins are indicated in the right lane. (b) Native-PAGE of wild-type mouse L-PGDS (lane 1) and the C65A mutant (lane 2) under nonreducing conditions. Bands of marker protein (pI = 4.4) are shown on both sides.
Mentions: The cDNAs of wild-type mouse L-PGDS without its N-terminal signal peptide of 24 amino acids and its C65A mutant were expressed in Escherichia coli BL21(DE3) (Invitrogen, CA, USA) as a glutathione (GSH) transferase-fusion protein. The fusion protein was purified by GSH-Sepharose 4B (GE Healthcare, Buckinghamshire, U.K.) column chromatography from a soluble fraction of E. coli cells. After incubation with thrombin (Sigma-Aldrich, WI, USA), L-PGDS protein was obtained and further purified by column chromatography on Superdex 75 (GE Healthcare) in 5 mM Tris-HCl pH 8.0, followed by Mono-S HR5/5 (GE Healthcare) chromatography with a linear gradient from 0 to 500 mM sodium chloride in 20 mM Tris-HCl, pH 7.5, at 293 K. Structural homogeneities of final purified preparations of the wild-type and C65A mutant of mouse L-PGDS were evaluated by SDS-PAGE (under reducing conditions) and by native PAGE (PAGE without a denaturing reagent, such as SDS, and under nonreducing conditions) as shown in Figure 1. The wild-type L-PGDS showed the same PGDS activity as that of the C89,189A mutant.(14) They were concentrated to 40 mg mL–1 in 100 mM HEPES-NaOH, pH 7.0, and 100 mM sodium chloride using a Centricon YM-10 membrane (10,000 nominal molecular weight limit; Millipore, MA, USA) and stored at 277 K. The protein concentration was determined spectrophotometrically at 280 nm.

Bottom Line: Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) catalyzes the isomerization of PGH(2) to PGD(2) and is involved in the regulation of pain and of nonrapid eye movement sleep and the differentiation of male genital organs and adipocytes, etc.Mouse L-PGDS with a C65A mutation was previously crystallized with citrate or malonate as a precipitant, and the X-ray crystallographic structure was determined at 2.0 Å resolution.The precipitant solution was 40% (w/v) PEG 8000, 100 mM sodium chloride, and 100 mM HEPES-NaOH (pH 7.0).

View Article: PubMed Central - PubMed

Affiliation: Maruwa Foods and Biosciences Inc. , 170-1 Tsutsui-cho, Yamatokoriyama, Nara 639-1123, Japan.

ABSTRACT
Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) catalyzes the isomerization of PGH(2) to PGD(2) and is involved in the regulation of pain and of nonrapid eye movement sleep and the differentiation of male genital organs and adipocytes, etc. L-PGDS is secreted into various body fluids and binds various lipophilic compounds with high affinities, acting also as an extracellular transporter. Mouse L-PGDS with a C65A mutation was previously crystallized with citrate or malonate as a precipitant, and the X-ray crystallographic structure was determined at 2.0 Å resolution. To obtain high-quality crystals, we tried, unsuccessfully, to crystallize the C65A mutant in microgravity under the same conditions used in the previous study. After further purifying the protein and changing the precipitant to polyethylene glycol (PEG) 8000, high-quality crystals were grown in microgravity. The precipitant solution was 40% (w/v) PEG 8000, 100 mM sodium chloride, and 100 mM HEPES-NaOH (pH 7.0). Crystals grew on board the International Space Station for 11 weeks in 2007, yielding single crystals of the wild-type L-PGDS and the C65A mutant, both of which diffracted at around 1.0 Å resolution. The crystal quality was markedly improved through the use of a high-viscosity precipitant solution in microgravity, in combination with the use of a highly purified protein.

No MeSH data available.


Related in: MedlinePlus