Codon-optimized (DNA 2.0) DNA constructs corresponding to the mature form of human LRPPRC (amino acid 60–1394) or SLIRP (18–109) were cloned in a pJexpress 401 (DNA 2.0) vector and the LRPPRC–SLIRP complex in pCDFDuet-1 (Novagen). LRPPRC harbours a 6×His fusion tag at the N-terminus in both vectors whereas SLIRP was His-tagged in the pJexpress 401 vector and untagged in pCDFDuet-1. The proteins were expressed in Rosetta 2 cells (EMD chemicals) by induction with 0.5 mM isopropyl-1-thio-β-D-galactopyranoside (IPTG) at 30°C for 16 h in Enpresso B media (Biosilta). After lysis, the proteins were purified over a His-Select Ni2+ (Sigma-Aldrich) resin and dialysed against H-0.2 (25 mM Tris–HCl [pH 7.8], 0.5 mM ethylenediaminetetraacetic acid (EDTA), 10% glycerol, 1 mM dithiothreitol, 200 mM NaCl) after the addition of TEV protease at a 1:50 protease:protein ratio. Further purification was conducted over a heparin column equilibrated in H-0.2. After washing with H-0.2 the proteins were eluted with H-0.6 and purified to homogeneity over a HiLoad 16/60 Superdex 200 pg gel filtration column (GE Healthcare) in buffer H-0.2 lacking glycerol.
Hiload 16 60 superdex 200 pg gel filtration column
Manufactured by GE Healthcare
Sourced in United Kingdom
The HiLoad 16/60 Superdex 200 pg gel filtration column is a size-exclusion chromatography column used for the separation and purification of proteins, peptides, and other biomolecules. The column features a prepacked matrix of cross-linked agarose and dextran that allows for efficient separation based on molecular size and shape.
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Lab products found in correlation
Magicmedia, by Thermo Fisher Scientific (4 mentions)
His select ni2 resin, by Merck Group (3 mentions)
His select ni2, by Merck Group (2 mentions)
Bl21 codonplus de3 ril competent cells, by Agilent Technologies (2 mentions)
Sp sepharose, by GE Healthcare (2 mentions)
Arctic express cells, by Agilent Technologies (2 mentions)
Desalting column, by GE Healthcare (1 mentions)
Ni2 nitrilotriacetate ni2 nta affinity column, by GE Healthcare (1 mentions)
Q sepharose column, by GE Healthcare (1 mentions)
Pet28a vector, by Thermo Fisher Scientific (1 mentions)
1 2 dipalmitoyl sn glycero 3 phosphocholine, by Avanti Polar Lipids (1 mentions)
1 2 dihexanoyl sn glycero 3 phosphocholine, by Avanti Polar Lipids (1 mentions)
Horseradish peroxidase, by Sangon (1 mentions)
Ni nta resin, by GE Healthcare (1 mentions)
Mosquito liquid handling robot, by SPT Labtech (1 mentions)
Sp650s, by Tosoh (1 mentions)
Kta start, by GE Healthcare (1 mentions)
Superdex 75 increase, by GE Healthcare (1 mentions)
Vivaspin concentrator, by Sartorius (1 mentions)
12 protocols using hiload 16 60 superdex 200 pg gel filtration column
1
Purification of LRPPRC-SLIRP Complex
2
Purification of HIF-2α-ARNT Complex
3
Purification of Human C6orf203 Protein
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A codon-optimized (Genscript) DNA construct corresponding to the mature form of human C6orf203 (amino acids 85–240) was cloned into a Fh8-Pet24d vector. Human C6orf203 was expressed in Arctic express cells (Agilent) at 16°C for 48 h in Magic Media (Thermo Fisher Scientific) with an approximate yield of 5 mg/l, assessed by Bradford protein assay. After lysis, the proteins were purified over a His-Select Ni2+ resin (Sigma-Aldrich) and dialyzed against H-0.2 (25 mM Tris-HCl (pH 7.4), 0.5 mM EDTA, 10% glycerol, 3 mM β-mercaptoethanol and 200 mM NaCl) after the addition of TEV protease at a 1:25 protease:protein ratio. The dialyzed protein was loaded on a second Ni2+ resin for TEV protease removal and the flow-through was collected. Further purification was conducted over a HiLoad 16/60 Superdex 200 pg gel filtration column (GE Healthcare) in buffer H-0.2 lacking glycerol with the addition of 2 mM dithiothreitol instead of β-mercaptoethanol. The purity was estimated at ∼95% following SDS-PAGE gel electrophoresis and staining with Coomassie (Supplementary Figure S1A ). RNA EMSA were performed as previously described (19 (link)).
4
Purification and Characterization of Mitochondrial Translation Initiation Factors
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A codon-optimized (Genscript) DNA construct corresponding to the mature form of human mtIF2 (amino acids 38–727) or human mtIF3 (amino acids 32–278) was cloned into a pET-24b vector (Novagen). Both constructs were expressed in Rosetta 2 cells (EMD chemicals) at 25 °C for 16 h in Magic Media (Thermo Fisher Scientific). After lysis, the proteins were purified over a His-Select Ni2+ resin (Sigma–Aldrich) and dialyzed against H-0.2 (25 mM Tris-HCl pH 7.4, 0.5 mM EDTA, 10% glycerol, 1 mM DTT, 200 mM NaCl). Further purification was conducted over a HiLoad 16/60 Superdex 200 pg gel filtration column (GE Healthcare) in buffer H-0.2 lacking glycerol.
The first 30 or 51 nucleotides of mitochondrially encoded cytochrome C oxidase 2 (MT-CO2) mRNA was purchased from Sigma–Aldrich or Eurofins, respectively.
The unlabeled E. coli, fMet-tRNAMeti was generously provided by Prof. Marina Rodnina, and the fMet-tRNAMeti labeled at the dihydrouridine residues with Cy3 by Prof. Barry S. Cooperman17 .
The first 30 or 51 nucleotides of mitochondrially encoded cytochrome C oxidase 2 (MT-CO2) mRNA was purchased from Sigma–Aldrich or Eurofins, respectively.
The unlabeled E. coli, fMet-tRNAMeti was generously provided by Prof. Marina Rodnina, and the fMet-tRNAMeti labeled at the dihydrouridine residues with Cy3 by Prof. Barry S. Cooperman17 .
5
Purification of Codon-Optimized ANGEL Proteins
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A codon-optimised (Genscript) DNA construct corresponding to DmANGEL (amino acids 26–354) was cloned into a Pet24b vector and HsANGEL2 (amino acids 165–544) or MmANGEL2 (amino acids 164–544) was cloned into a Fh8-Pet24d vector. HsANGEL2 and MmANGEL2 were expressed in Arctic express cells (Agilent) at 16 °C for 48 h in Magic Media (Thermo Fisher Scientific). After lysis, the proteins were purified over a His-Select Ni2+ resin (Sigma-Aldrich) and dialysed against H-0.5 (25 mM Tris pH 7.4, 0.5 mM EDTA, 10% glycerol, 3 mM β-mercaptoethanol, 500 mM NaCl) after the addition of TEV protease at a 1:50 protease:protein ratio. The dialysed proteins were loaded on a second Ni2+ resin for TEV protease removal and the flow through was collected. Further purification was conducted over a HiLoad 16/60 Superdex 200 pg gel filtration column (GE Healthcare) in buffer H-0.5 lacking glycerol with the addition of 2 mM dithiothreitol instead of β-mercaptoethanol. DmANGEL was expressed and purified analogous to HsANGEL2 except that no His tag removal was performed.
6
Phospholipid Characterization and Enzyme Purification
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PC (purity 95%, from soybean), 1, 2-dihexanoyl-sn-glycero-3-phosphocholine (6:0/6:0-PC) (purity > 99%), 1, 2-dioctanoyl-sn-glycero-3-phosphocholine (8:0/8:0-PC) (purity > 99%), 1, 2-dilauroyl-sn-glycero-3-phosphocholine (12:0/12:0-PC) (purity > 99%), 1, 2-dimyristoyl-sn-glycero-3-phosphocholine (14:0/14:0-PC) (purity > 99%), 1, 2-di-palmitoyl-sn-glycero-3-phosphocholine (16:0/16:0-PC) (purity > 99%) and 1, 2-distearoyl-sn-glycerol-3-phosphocholine (18:0/18:0-PC) (purity > 99%) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). PS standard (purity > 97%) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Choline oxidase was prepared by the previously reported method [40 (link)]. Horseradish peroxidase was purchased from Sangon Biotech Co., Ltd. (Shanghai, China). Hypersil GOLD Silica column (Dim. 4.6 × 250 mm, particle size 5.0 µm) was obtained from Thermo Fisher Scientific (Waltham, MA, USA). Escherichia coli SHuffle T7 Express competent cell were purchased from New England BioLabs (Beijing, China). The plasmid pET28a vector was purchased from Invitrogen (Carlsbad, CA, USA). Ni2+-nitrilotriacetate (Ni2+-NTA) affinity column, desalting column, Q-Sepharose column and Hiload 16/60 Superdex 200 pg gel filtration column were obtained from GE Healthcare Life Sciences (Pittsburgh, PA, USA). All other chemicals used in the present study were analytical grade.
7
Hsc70 Recombinant Protein Production
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Human T199A Hsc701–646 (UniProt ID P11142, referred to as Hsc70 in this manuscript) was expressed in E. coli BL21-CodonPlus (DE3)-RIPL cells from a pProEx plasmid carrying an N-terminal polyhistidine tag (a gift from J. Young, McGill University, Montreal, Canada). Cells were grown in minimal M9 D2O media containing 1 g/L 15NH4Cl and 3 g/L [2H,12C]-glucose as the sole nitrogen and carbon sources respectively to an OD600 of ∼0.8, at which point expression was induced by the addition of 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and allowed to proceed overnight at 25°C. Following expression, bacteria were harvested and the Hsc70 protein purified on nickel nitrilotriacetic acid (Ni-NTA) resin (GE Healthcare) under denaturing conditions (6 M GuHCl) to ensure complete removal of bound nucleotides. Hsc70 was refolded on the Ni-NTA column by gradually reducing the concentration of denaturing agent in the wash buffer (from 6 M to 0 M GuHCl), and the N-terminal polyhistidine tag was removed via TEV (tobacco etch virus) protease cleavage. The cleaved protein was further purified on a HiLoad 16/60 Superdex 200 pg gel filtration column (GE Healthcare) equilibrated with 50 mM HEPES (pH 7.5), 50 mM KCl, 1 mM dithiothreitol (DTT), and 0.03% NaN3.
8
Purification of HIF-2α-ARNT Complex
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To obtain HIF-2α-ARNT complex proteins, the pSJ2-HIF-2α plasmid was co-transformed along with pMKH-ARNT into BL21-CodonPlus (DE3)-RIL competent cells (Agilent Technologies). Following 0.1 mM IPTG induced protein expression overnight at 16 °C, cell pellets were lysed by sonication, and supernatants were applied onto pre-packed His∙Bind resin (Novagen). The bound proteins were further purified using SP Sepharose (GE Healthcare), and the eluted fractions were then loaded on a HiLoad 16/60 Superdex 200pg gel filtration column (GE Healthcare) equilibrated in 20 mM Tris (pH 8.0) and 400 mM NaCl. DTT was added to the pooled protein peak fractions at 10 mM. The heterodimeric proteins of HIF-2α and ARNT-GFP were prepared similarly as described above, except that the pMKH-ARNT-GFP plasmid was used in the place of pMKH-ARNT. The ARNT-GFP protein was co-expressed and purified in complex with HIF-1α and NPAS3 (plasmids made in our previous studies3 (link),8 (link)), respectively. The single PAS-B domain of HIF-2α was produced by transformation of pSJ2-HIF-2α (241–361) into BL21-CodonPlus (DE3)-RIL, followed by overnight expression, and purification using His-tag affinity chromatography and gel filtration chromatography.
9
BRG Fab-RNA Complex Crystallization
10
Purification and Analysis of C. botulinum Toxin
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C. botulinum serotype D strain 4947 was cultured anaerobically for 5 days using dialysis [13] (link). Crude TC was precipitated with 60% (w/v) saturated ammonium sulfate, dialyzed against 50 mM acetate buffer (pH 4.0) containing 0.2 M NaCl, and applied to a TOYOPEARL SP-650S (Tosoh, Tokyo, Japan) cation-exchange column (1.6 cm×40 cm) equilibrated with dialysis buffer. TC species bound to the resin were eluted with a linear gradient of NaCl (0.2–0.8 M). Each fraction (M-TC, M-TC/HA-70, one-arm L-TC, two-arm L-TC, and three-arm L-TC) was pooled separately, concentrated, and further purified with a HiLoad 16/60 Superdex 200 pg gel-filtration column (GE Healthcare, Little Chalfont, UK 1.6 cm×60 cm) equilibrated with 50 mM acetate buffer (pH 5.0) containing 0.15 M NaCl. The TC fraction was then applied to a Mono S HR5/5 cation-exchange column (GE Healthcare UK; 0.5 cm×5 cm) equilibrated with 50 mM acetate buffer (pH 5.0) and eluted using a linear gradient of NaCl (0–0.5 M). The purities of the M-TC, M-TC/HA-70, one-arm L-TC, two-arm L-TC, and three-arm L-TC were evaluated by native PAGE and SDS-PAGE.
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