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49 protocols using EmulsiFlex-C5 homogenizer

1

Isotopically Labeled Protein Expression and Purification

Labeled eNP proteins (15N-, 13C, 2H) were expressed as cleavable fusions to maltose binding protein (MBP) in BL21(DE3) E. coli cells (Novagen). For labeling, M9 2H2O medium was used supplemented with 1.5 g/L 15NH4Cl, 2g/L D-glucose (13C6,1,2,3,4,5,6,6-d7), 0.1 mM CaCl2, 1 mM MgSO4, and 1X vitamin mix (Sigma-Aldrich B6891). Protein expression was induced at an optical density of 0.6 with 0.5 mM IPTG, and cells were grown for 12‒15 h at 18 °C. Cells were harvested, resuspended in lysis buffer containing 25 mM Tris pH 7.5, 150 mM NaCl, 20 mM imidazole, and 5 mM 2-mercaptoethanol (BME), lysed using an EmulsiFlex-C5 homogenizer (Avestin), and clarified by centrifugation at 30,000 x g at 4 °C for 45 min. eNP proteins were purified using a series of affinity and ion exchange chromatographic columns. TEV protease was added to cleave the MBP tag prior to a final purification on a size exclusion column. The purity of the samples was determined by SDS-PAGE analysis.
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2

Recombinant Expression and Purification of β2-Microglobulin

Wild-type β2m was expressed recombinantly in Escherichia coli BL21(DE3) pLysS cells by induction with
1 mM IPTG overnight at 37 °C, following methods described previously.39 (link) Cells were lysed in 25 mM Tris-HCl buffer, pH
8.0 and with an Avestin Emulsiflex-C5 homogenizer. β2m is accumulated in inclusion bodies. To extract the β2m from inclusion bodies, the cell pellet was washed five times
with 25 mM Tris-HCl buffer, pH 8.0 and solubilized in 25 mM Tris-HCl,
pH 8.0 buffer containing 8 M urea, rocking overnight at room temperature.
The protein was verified to be in the soluble fraction by SDS-PAGE.
β2m was refolded by dialyzing against 25 mM Tris-HCl
buffer, pH 8.0 at 4 °C and purified by anion exchange (HiTrap
Q HP, GE Healthcare). The protein was further purified by size exclusion
chromatography with a HiLoad 26/600 Superdex 75 gel filtration column
(GE Life Sciences). Protein purity was verified by SDS-PAGE, and concentrations
for experiments were determined by measuring the absorbance at 280
nm using a molar extinction coefficient of 19,060 M–1 cm–1. [U–15N]-enriched β2m was expressed recombinantly for NMR using the same protocol
in HCDM1 minimal media supplemented with 15N-ammonium chloride.
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Mutant DNAs were generated by PCR-based mutagenesis using the Quickchange mutagenesis kit (Stratagene), or were purchased at GenTech. All constructs were verified by sequencing. BFL1∆C (lacking the C-terminal 24 residues) and its variants were expressed in Escherichia coli BL21 (DE3) using the pNIC28-Bsa4 vector (New England Biolabs). Cells were induced at OD of 1.5–2 with 1 mM IPTG, and grown overnight at 18°C. The harvested cells were lysed at 4°C with a Emulsiflex C5 homogenizer (Avestin) in 500 mM NaCl, 20 mM Imidazole, 50 mM Na2HPO4 pH 7.5, 1 mM TCEP, 10% glycerol, 1 mg/ml lysozyme, 2.5 µg/ml DNAse I, and complete EDTA-free protease inhibitor cocktail tablets (Roche). BFL1∆C proteins were purified from the supernatant by nickel-affinity and size-exclusion chromatography and stored in 100 mM KCl, 10 mM Hepes, pH 7.4, 1 mM EDTA (KHE buffer) supplemented with 1 mM TCEP and 10% w/v glycerol. BAX, cBID, BCLXL, BCLXLΔC, and their variants were expressed and purified as described [29 (link)]. All protein preparations were >95% pure as assessed by SDS-PAGE and Coomassie staining. In a typical protein labeling reaction, Alexa 488, Alexa 647 or NBD were incubated with BCL2 proteins at a molar ratio of 10:1, samples were incubated overnight at 4°C, and subsequently eluted over a PD-10 column equilibrated with KHE.
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LDE-MTX was prepared from a lipid mixture composed of 100 mg cholesteryl oleate, 200 mg egg phosphatidylcholine (Lipoid, Germany), 10 mg triglycerides, 12 mg cholesterol and 60 mg of MTX (5 (link),19 (link)). The aqueous phase (100 mg of polysorbate 80 and 10 mL of Tris-HCl buffer, pH 8.05) was kept at room temperature. The pre-emulsion was obtained adding the hydrophilic phase to the oil phase by ultrasonic radiation until complete dissolution of the drug. Emulsification of the compounds was obtained by high-pressure homogenization using an Emulsiflex C5 homogenizer (Avestin, Canada). After homogenization at constant temperature, the nanoemulsion was centrifuged at 1800 g for 15 min at 4°C to separate the emulsified from unbound MTX. The nanoemulsion was sterilized by passage through 0.22 μm pore filter (Millipore, USA) and kept at 4°C until it was used. The association of MTX to LDE was measured by HPLC before the treatment (22 (link)).
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Plasmids encoding SFTSV L 1–231 aa, HRTV L 1–231 aa, and IAV PA 1–192 aa were transformed in BL21(DE3) E. coli cells (Novagen), cultured in Luria Broth media or in M9 medium (for selenomethionine labeled proteins) at 37°C, induced with 0.5 mM IPTG, and grown for 12 hr at 15°C. Cells were harvested and resuspended in lysis buffer containing 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 20 mM imidazole, 5 mM 2-mercaptoethanol, and were lysed using an EmulsiFlex-C5 homogenizer (Avestin). Lysates were clarified by centrifugation at 47,000 × g at 4°C for 40 min. Proteins were purified using a series of chromatographic columns and a size exclusion column as a final step. Protein purity was determined by Coomassie staining of SDS-PAGE.
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SARS-CoV-2 N constructs were expressed as His-tag fusion proteins in BL21 (DE3) E. coli cells (Novagen). At OD600 of 0.6–0.7, recombinant protein expression was induced with 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) for 12–14 h at 18°C. Cells were harvested and resuspended in lysis buffer containing 20 mM Tris (pH 7.5), 1 M NaCl, 20 mM imidazole, 5 mM 2-mecaptoethanol (BME). Cells were lysed using an EmulsiFlex-C5 homogenizer (Avestin) and lysates were clarified by centrifugation at 30,000 x g at 4 °C for 40 min. N proteins were purified using affinity tag and gel filtration columns. Purity of N proteins were determined by Coomassie staining of SDS-PAGE.
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Fluorescent proteins were produced using pROD93 expression vector in BL21(DE3) strains. Cells were grown at 37°C until they reached an OD600 ~ 0.6. Protein expression was then induced by adding 0.2% of L-arabinose and incubating the culture at 30°C. After 4 hours of induction, bacteria were centrifuged and the pellet was resuspended in Resuspension Buffer (25 mM Tris HCl pH 7.5, 250 mM NaCl) supplemented with one tablet of cOmplete protease inhibitor (Roche, Cat # 04693159001). Cells were then lyzed using a high-pressure Emulsiflex C5 homogenizer (Avestin), and the lysate was cleared by ultra-centrifugation (35 000 rpm, 1 hour, 4°C). The lysate was added to the Ni-IDA resin (Takara, Cat # 635660) equilibrated with Binding Buffer (50 mM NaPi, 20 mM Imidazole, 300 mM NaCl, 10% glycerol, pH 8) and incubated for at least an hour at 4°C. The resin was transferred to a disposable column and washed out at least 5 times with Binding Buffer and twice with Binding Buffer that has a higher concentration of imidazole (40 mM). Protein was eluted in Elution Buffer (50 mM NaPi, 200 mM Imidazole, 300 mM NaCl, 10% glycerol, pH 8). The protein was then dialyzed in Slide-A-Lyzer MINI Dialysis Devices immersed in the Storage Buffer (25 mM Tris HCl pH8, 300 mM NaCl, 10% glycerol) and finally stored, protected from light, at −20°C.
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The LDE-paclitaxel oleate preparation was made according to the methods described in
previous studies (11 (link),27 (link),28 (link)). Briefly, a lipid
mixture composed by 135 mg cholesteryl oleate (Alfa Aesar, USA), 333 mg egg
phosphatidylcholine (Lipoid, Germany), 132 mg miglyol 812N (Sasol Germany GmbH,
Germany), 6 mg cholesterol (Fabrichem, USA) and 60 mg of paclitaxel oleate
(Pharmaceuticals Co., China), was added to an aqueous phase consisting of 100 mg of
polysorbate 80 (Merck, Germany) and 10 mL Tris-HCl buffer, pH 8.05. A pre-emulsion
was obtained by ultrasonic radiation until complete solubilization of the drug.
Emulsification of all lipids, drug and aqueous phase was obtained by high-pressure
homogenization using an Emulsiflex C5 homogenizer (Avestin, Canada). After
homogenization cycles, the formed emulsion was centrifuged and the nanoparticle
sterilized by passage through 0.22 μm pore polycarbonate filter (Millipore, USA) and
kept at 4°C until used.
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Genomic DNA was isolated using a Wizard Genomic DNA purification kit (Promega) according to the manufacturer's instructions. fhbp was amplified from N. cinerea genomic DNA using primers Ffhbp (5′-GCCATATGATGGCCGCCGACAT-3′; NdeI site underlined) and Rfhbp (5′-GCCTCGAGTTGCTTGGCGGCAAGGCCGATAT-3′; XhoI site underlined). The product was ligated into pET-21b (Table 2), resulting in a construct for expression of fHbp with a C-terminal His tag under an inducible promoter. fHbp expression was performed as described previously (44 (link)).
In brief, for protein expression, E. coli BL21(D3) (Agilent) containing fHbp expression constructs was grown in liquid medium to an A600 of 0.4 to 0.8; then, IPTG was added to a final concentration of 1 mM. After 4 h, the bacteria were harvested by centrifugation at 5,000 × g for 30 min at 4°C prior to cell lysis in an EmulsiFlex-C5 homogenizer (Avestin) at 15,000 lb/in2. The lysates were centrifuged at 50,000 × g for 30 min at 4°C, and recombinant fHbp was purified by affinity chromatography using a HisTrap column (GE Healthcare) and eluted with 200 mM imidazole. Further purification was performed with an AKTA purifier (GE Healthcare) by anion-exchange chromatography (HiTrapQ HP column; GE Healthcare). Protein concentrations were estimated using a Nanodrop 2000c spectrophotometer (Thermo Scientific).
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Heterotrimeric human kinase dead AMPK His6-α1(D141A)β1γ1 was expressed in E. coli strain Rosetta (DE3) using the pET DUET expression system (Novagen) as described previously40 (link). Briefly, α1(D141A) and γ1, cloned into pET DUET, were co-expressed with β1, cloned into pET RSF DUET. AMPK stoichiometrically myristoylated on the β subunit residue Gly2 was generated by co-expression with N-myristoyltransferase inserted into pET RSF DUET MCS2 (BglII/XhoI)15 . Expression cultures were grown in Luria Bertani broth and induced at 16 °C with 0.25 mM isopropyl β-D-thiogalactopyranoside, prior to overnight incubation. Cells were lysed in 50 mM Tris.HCl, pH 7.8, 150 mM NaCl, 10% glycerol, 50 mM imidazole, 2 mM β-mercaptoethanol, 0.1 mM Leupeptin, 0.1 mM AEBSF and 1 mM Benzamidine HCL using a pre-cooled EmulsiFlex-C5 homogenizer (Avestin). AMPK purified using nickel Sepharose and size exclusion chromatography (HiLoad 16/60 Superdex 200 PG). Final storage buffer consisted of 50 mM Tris.HCl, pH 7.8, 150 mM NaCl, 10% glycerol and 2 mM TCEP. All preparations were verified by TOF mass spectrometry.
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