Production and conjugation DUPA-CD3 conjugates using oxime ligation was carried out as previously described in (10 (link)) and (51 (link)) and as depicted in fig. S2. Briefly, anti-CD3 Fab sequences inserted 3′ of a STII leader peptide in a vector that harbors the suppressor transfer RNA (tRNA) E9RS aminoacyl-tRNA synthase. To enable site-specific unnatural amino acid incorporation for DUPA conjugation, the following residues were mutated to the TAG amber nonsense codon, where indicated (UCHT1 at HC residue K138 and/or LC residue S202; huL5H2-based conjugates at HC residue K141 or LC residue S205). Production of Fabs was carried out in Escherichia coli, with media containing the unnatural amino acid p-acetylphenylalanine. Fabs were purified using CaptureSelect IgG-CH1 affinity matrix (Thermo Fisher Scientific) and eluted using low-pH buffer. Conjugation was carried out with similar conditions as previously described (10 (link)), using 30 to 40 molar excess of DUPA-linker in 50 mM sodium acetate buffer (pH 4.5). Conjugations were carried out for 24 to 48 hours. Subsequent buffer exchange and polishing by strong cation exchanger resin SP Sepharose Fast Flow (GE Healthcare Life Sciences) were performed to remove free, nonconjugated DUPA-linker.
Sp sepharose fast flow
Manufactured by GE Healthcare
Sourced in United States, Sweden
SP Sepharose Fast Flow is a strong cation exchange chromatography resin. It is designed for the purification of proteins, peptides, and other biomolecules. The resin features a rigid, highly cross-linked agarose matrix with sulfopropyl (SP) functional groups, providing a high binding capacity and fast flow rates.
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Lab products found in correlation
Captureselect igg ch1 affinity matrix, by Thermo Fisher Scientific (1 mentions)
Amylose resin beads, by New England Biolabs (1 mentions)
Bl21 codonplus de3 rp, by Agilent Technologies (1 mentions)
Hiload 26 600 superdex200 pg, by GE Healthcare (1 mentions)
Bovine serum albumin (bsa), by Bio-Rad (1 mentions)
Nupage system, by Thermo Fisher Scientific (1 mentions)
Ferrocene, by Merck Group (1 mentions)
Collagen 1 from rat tail, by Thermo Fisher Scientific (1 mentions)
Hydrogen peroxide 30, by Thermo Fisher Scientific (1 mentions)
Polisher millipore ultra purification system, by Merck Group (1 mentions)
Protector rnase inhibitor, by Merck Group (1 mentions)
Dual hplc, by Biosearch Technologies (1 mentions)
Centrifugal filter device, by Merck Group (1 mentions)
Amicon ultra, by Merck Group (1 mentions)
Akta system, by GE Healthcare (1 mentions)
Isopropyl β d thiogalactopyranoside iptg, by Sangon (1 mentions)
O nitrophenyl β d galactopyranoside onpg, by Sangon (1 mentions)
Bca assay kit, by Beyotime (1 mentions)
Polypeptone, by Fujifilm (1 mentions)
Other analytical grade reagents, by Sinopharm (1 mentions)
30 protocols using sp sepharose fast flow
1
DUPA-CD3 Conjugate Production and Characterization
2
Purification of SARS-CoV-2 Nucleocapsid Protein
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A pET21b vector containing the SARS-CoV-2 N gene (amino acids 1–419) was kindly provided by Prof. Yoshiharu Matsuura from Research Institute for Microbial Diseases, Osaka University. An additional HRV-3 C restriction site was added into the N gene fragment, and the fragment was then subcloned into a pMAL-c2 vector [29] (link), [30] (link). The recombinant plasmid was propagated and subsequently transformed into competent Escherichia coli strain BL21 for protein expression.
E. coli BL21 containing the recombinant plasmid was shake-flask cultured (200 rotation per minute at 37 °C) in Luria Bertani broth supplemented with 50 μg/ml carboxy-benzylpenicillin as a selective agent. Isopropyl β-d-1-thiogalactopyranoside was added when the culture had reached an absorbance of 0.6–0.7 at 600 nm. The suspension was further cultured at 32 °C for 3 h, or 19 °C overnight. The cell pellet was concentrated and lysed to release the expressed protein into the supernatant. The supernatant was then separated from the pellet. The maltose-binding protein (MBP)-bound N protein was purified from the supernatant using amylose resin beads (NEB); the eluted N protein was then cleaved from MBP using HRV-3 C protease. The N protein was purified by cation exchange using SP Sepharose Fast Flow (GE Healthcare), and the purified N protein was confirmed by SDS-PAGE.
E. coli BL21 containing the recombinant plasmid was shake-flask cultured (200 rotation per minute at 37 °C) in Luria Bertani broth supplemented with 50 μg/ml carboxy-benzylpenicillin as a selective agent. Isopropyl β-d-1-thiogalactopyranoside was added when the culture had reached an absorbance of 0.6–0.7 at 600 nm. The suspension was further cultured at 32 °C for 3 h, or 19 °C overnight. The cell pellet was concentrated and lysed to release the expressed protein into the supernatant. The supernatant was then separated from the pellet. The maltose-binding protein (MBP)-bound N protein was purified from the supernatant using amylose resin beads (NEB); the eluted N protein was then cleaved from MBP using HRV-3 C protease. The N protein was purified by cation exchange using SP Sepharose Fast Flow (GE Healthcare), and the purified N protein was confirmed by SDS-PAGE.
3
Purification of Recombinant Tau and α-Synuclein
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Escherichia coli BL21-CodonPlus (DE3)-RP (Agilent) was transformed with a pET28a plasmid encoding WT or P301L tau (full-length 0N4R). Terrific broth cultures (1 l) supplemented with 50 mg/l kanamycin or 50 mg/l chloramphenicol were inoculated with 20 ml of starter cultures and grown for 8 h. The cultures were induced with 1 mM IPTG and grown for another 16 h. Cells were harvested and resuspended in 50 ml/l 20 mM MES, pH 6.8, 1 mM EGTA, 1 mM magnesium chloride, 5 mM DTT, and 1 cOmplete protease inhibitor cocktail (Roche) followed by microfluidizer lysis. The lysates were boiled for 20 min and centrifuged at 48,400g. The cleared lysates were applied to a cation exchange column (SP Sepharose Fast Flow; GE Healthcare), and fractions were eluted with a sodium chloride gradient. Fractions containing 0N4R tau were applied to a reversed-phase HPLC column and eluted with an acetonitrile gradient (1%/min) + 0.1% TFA gradient; the peak fractions were then lyophilized. The lyophilizates were dissolved in PBS + 1 mM DTT and purified by size-exclusion chromatography (HiLoad 26/600 Superdex 200 pg; GE Healthcare). Peak fractions were analyzed by SDS-PAGE, and fractions containing <95% 0N4R tau were pooled, snap-frozen, and stored at −80 °C. Recombinant full-length α-synuclein and tau repeat domain (K18) were expressed and purified as previously described (72 (link), 73 (link)).
4
Recombinant Kai Protein Expression and Purification
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Recombinant Kai proteins (i.e. KaiA, KaiB, and KaiC) of the cyanobacterium Synechococcus elongatus PCC 7942 were synthesized using the conventional E. coli expression system, as reported previously28 (link). The expression vector for the N-terminal domain fragment (CI-model, residues 2-247) of KaiC was generated from that of wild-type KaiC29 (link). Expression vectors of KaiC-variants, E77Q/E78Q, R393C, and F470Y, were generated via the site-directed mutagenesis protocol28 (link). Proteins were purified using a Strep-tactin Sepharose column (IBA GmbH, Göttingen, Germany), followed by liquid chromatography with SP Sepharose Fast Flow and superdex 75 PG 26/60 columns (GE Healthcare Co. Chicago, IL, USA). Protein concentration was determined via the Bradford method, using protein assay kits (FUJIFILM Wako Pure Co., Osaka, Japan) with bovine serum albumin (Bio-Rad Laboratories, Inc., Hercules, CA, USA) as the standard.
5
Purification of Recombinant RGS8-His Protein
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RGS8-His was expressed in E coli RosettaBlue (DE3)pLacI (Merck, Germany) as described previously.11 (link) For protein purification, bacterial sediments were resuspended in 50 mM TRIS-HCl pH 8.0, 0.3 M NaCl, 1 mM phenylmethylsulfonyl fluoride, 25 mM dithiothreitol, and homogenized with PandaPLUS 2000 laboratory high-pressure homogenizer (GEA, Germany). Lysed bacteria were centrifuged at 21,200g for 30 minutes at 4°C. Pellets were solubilized in 10 mM TRIS-HCl pH 8.0, 0.3 M NaCl, 8 M urea, 0.5 mM dithiothreitol, and 20 mM imidazole for 30 minutes, followed by centrifugation at 21,200g for 30 minutes at 4°C. RGS8-His was purified from the supernatant by immobilized metal chelate affinity chromatography using Nickel Rapid Run resin (ABT, Spain) and ion-exchange chromatography (SP Sepharose Fast Flow, GE Healthcare, US), eluted in 50 mM sodium phosphate pH 7.4, 8 M urea, 1,000 mM NaCl. Protein analysis was performed by SDS-PAGE using the NuPAGE system according to the manufacturer's manual (Invitrogen) and by mass spectrometry.
6
Ion Exchange Chromatography for Protein Purification
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The CEC was carried out using different volumes of SP-Sepharose Fast Flow (GE Healthcare/Cytiva, UK) resin packed in columns from different sizes attached to Äkta Avant 150. The volumetric flow for all purifications was set at 2.26 mL/min·cm2. Two strategies were tested in this purification step. In the first strategy, the system was equilibrated with 5 CV of 25 mM sodium acetate buffer with 300 mM NaCl, pH 4.0. The elution was performed by a discontinuous gradient of NaCl at 500, 800, and 1000 mM in 25 mM acetate buffer, pH 4.0. In the second strategy, the system was equilibrated with 5 CV of 50 mM sodium acetate buffer, pH 5.5 or 6.0. The pH and conductivity of the fraction recovered from the previous step was corrected to match both the pH and conductivity of the equilibration buffer. After loading all the sample, the elution was performed by a linear gradient from 0 to 1000 mM of NaCl. In both strategies, the volume of buffer used in each step of the elution was 5 CV for the discontinuous gradient and 20 CV for the linear gradient.
7
Purification of HIF1A and Hph-1-G4D
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HIF1A was generated and Hph-1-G4D (GAL4-DBD) was purified as described by [18 (link)]. In brief, Homo sapiens HIF1A (NCBI Reference Sequence: NM_001530.3) was amplified using the polymerase chain reaction (PCR). The PCR product was inserted into the pEGFPN1 plasmid vector (Invitrogen, Carlsbad, CA, USA) using restriction enzyme NheI (Takara Bio, Otsu, Japan) at 5′ termini and KpnI (Takara Bio) at 3′ termini of the PCR fragment. The DNA of G4D combined with Hph-1 was transformed with Escherichia coli BL-21 Star (DE3) pLysS (Invitrogen). The recombinant proteins were subsequently mixed with SP Sepharose Fast Flow (GE Healthcare, Milwaukee, WI, USA) and Hph-1-G4D protein was eluted. The eluted proteins were desalted using PD-10 Sephadox B-25 (Amersham Pharmacia Biotech, Piscataway, NJ, USA) with 10% glycerol phosphate-buffered saline (Sigma-Aldrich, St Louis, MO, USA).
8
Aminoglycoside Aptamer Purification and Characterization
9
Enzyme Purification via SP Sepharose
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The retentate from the ultrafiltration was purified using the SP Sepharose Fast Flow (GE Healthcare Life Sciences) resin in a Äkta purifier chromatography system. Equilibration buffer (Buffer A or A1) was 0.1 M or 0.01 M sodium acetate/ acetic acid buffer (pH 5.0). Elution buffer (Buffer B) contained 1 M NaCl, 100 mM Tris and 10 mM EDTA (pH 7). The enzyme was eluted using a salt gradient of 0 to 100 % Buffer B over 1 h or through step-wise increases to 25 %, 50 and 100 % Buffer B. Cleaning-in-Place was done using 0.5 M NaOH through the A2 inlet of pump A.
10
Purification of Recombinant Protein SCI-57
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Analysis was carried out with a column of SP-Sepharose Fast Flow (GE Healthcare, Sweden) connected to the FPLC equipment (ÄKTA avant 25, Amersham Biosciences, USA). Recombinant human insulin (PISA pharmaceutical, México) on citrate buffer was used as positive control (1.75 mg/ml). The column was equilibrated with 10 ml of binding buffer (100 mM citrate buffer pH 4.3). Then, 5 ml of NLE or insulin solution was passed through the column. The column was washed with 5 ml of washing buffer (100 mM citrate buffer pH 4.3). The mobile phases were buffer A (100 mM citrate buffer pH 4.3) and buffer B (100 mM citrate buffer pH 4.3 with 1M NaCl). The elution profile to achieve the separation constituted a gradient of 100% B for 60 min with a continuous flow rate of 0.4 ml/min; samples were collected in 1 ml fractions. Elution fractions were monitored by a single path ultraviolet monitor at 280 nm. SCI-57 presence on the fractions was evaluated by 13% Tricine SDS-PAGE.
The fractions containing SCI-57 were filtrated through centrifugal filter devices with a cutoff of 3 kDa (Millipore, USA).
The fractions containing SCI-57 were filtrated through centrifugal filter devices with a cutoff of 3 kDa (Millipore, USA).
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