A 90 amino acid fragment of human-derived KEX1 was cloned into the pET28b(+) expression vector (GenScript) in Escherichia coli BL21(DE3) pLysS (Thermo Fisher Scientific) and used to produce an approximately 11 kDa recombinant protein. Microtitre plates (Immunolon 4HBX; Thermo Fisher Scientific) were coated with 5 µg/mL of purified KEX1 in phosphate-buffered saline (PBS). Heat-inactivated plasma was diluted 1:100 in blocking buffer (PBS with 5% non-fat milk); 50 µL of plasma were plated into KEX1-coated wells, and serial dilutions were made to determine end point titres. Goat antihuman immunoglobulin-conjugated horseradish peroxidase (1:10 000, IgG; Sigma-Aldrich) was used for detection, and plates were developed by standard methods. Plasma samples from this cohort that had an undetectable Pc antibody titre were used as negative controls. The reciprocal end point titre (RET) was calculated as the highest dilution at which the optical density was the same or less than that of the negative control. To determine whether patients with low anti-KEX1 antibody titres had a generalised defect in humoral immunity, tetanus toxoid antibody levels were determined using commercial ELISA kit (MyBioSource).
Escherichia coli bl21 de3 plyss
Manufactured by Thermo Fisher Scientific
Sourced in United States, Japan
Escherichia coli BL21(DE3) pLysS is a bacterial strain commonly used in protein expression studies. It is a genetically modified variant of the BL21(DE3) strain, which is designed for efficient expression of recombinant proteins. The pLysS plasmid in this strain provides additional control over protein expression by suppressing basal-level expression of the target protein.
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Lab products found in correlation
T4 dna ligase, by Thermo Fisher Scientific (2 mentions)
Prset emgfp bacterial expression vector, by Thermo Fisher Scientific (2 mentions)
Bacto yeast extract, by BD (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Pet 28b expression vector, by GenScript (1 mentions)
Immunolon 4hbx, by Thermo Fisher Scientific (1 mentions)
Pierce bicinchoninic acid bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Glutathione agarose resin, by Thermo Fisher Scientific (1 mentions)
E coli bl21 de3 plyss, by Thermo Fisher Scientific (1 mentions)
Ni nta resin, by Qiagen (1 mentions)
Bicinchoninic acid kit, by Merck Group (1 mentions)
Isopropyl β d thiogalactopyranoside, by Merck Group (1 mentions)
Phusion dna polymerase, by Thermo Fisher Scientific (1 mentions)
Qiaprep spin miniprep kit, by Qiagen (1 mentions)
Restriction endonuclease, by New England Biolabs (1 mentions)
Ni nta agarose bead, by Qiagen (1 mentions)
Ni nta agarose, by Qiagen (1 mentions)
Complete protease inhibitor cocktail tablet, by Roche (1 mentions)
Bacto tryptone, by BD (1 mentions)
Prsetb, by Thermo Fisher Scientific (1 mentions)
17 protocols using escherichia coli bl21 de3 plyss
1
Quantifying Anti-KEX1 Antibodies in Plasma
2
Purification of SARS-CoV-2 Viral Proteins
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Recombinant proteins were expressed and purified using the methods described previously (35 (link)). Briefly, His-tagged nsp1β and the PLP2 domain of nsp2 were expressed in Escherichia coli BL21(DE3)pLysS (Thermo Fisher Scientific, Waltham, MA) and purified using Ni-nitrilotriacetic acid agarose resin (Ni-NTA; Qiagen, Germantown, MD). GST-tagged nsp1β and mutants thereof were expressed in E. coli BL21(DE3)pLysS (Thermo Fisher Scientific, Waltham, MA) and purified using glutathione agarose resin (Thermo Fisher Scientific, Waltham, MA). His-tagged PCBP2 was expressed in E. coli BL21(DE3)pLysS (Thermo Fisher Scientific, Waltham, MA) and purified using Ni-NTA resin (Qiagen, Germantown, MD). Proteins were dialyzed, quantified with the Pierce bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, Waltham, MA), and stored at −80°C.
3
Bacillus subtilis CH11 Isolation and Characterization
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Bacillus subtilis CH11 strain isolated from Chilca Salterns in Lima, Peru, belonged to the collection of the Molecular Biology Laboratory, Faculty of Pharmacy and Biochemistry, Universidad Nacional Mayor de San Marcos. This strain was conserved at -80 • C in TSB medium/glycerol 30% (v/v). TSB medium (g/L): casein peptone, 17; K 2 HPO 4 , 2.5; glucose, 2.5; NaCl, 5; soya peptone, 3; pH 7.3. LB-Miller (g/L): yeast extract, 5; peptone from casein, 10; NaCl, 10; pH 7.0. T4 DNA ligase, Phusion DNA polymerase, and Escherichia coli BL21(DE3)pLysS were purchased from Thermo Scientific ® (Waltham, MA, USA). Restriction endonucleases were obtained from New England Biolabs ® (Ipswich, MA, USA). The QIAprep ® Spin Miniprep Kit was acquired from QIAGEN (Hilden, Germany). pET-15b and BugBuster ® Master Mix were from Novagen ® (Merck-Darmstadt, Germany). Finally, the Bicinchoninic Acid Kit and Isopropyl β-D-thiogalactopyranoside were procured from Sigma-Aldrich ® (St. Louis, MO, USA).
4
Expression and Purification of Recombinant Lci2
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His-tagged recombinant Lci2 (originally named Lci2B) was expressed in Escherichia coli BL21(DE3) pLysS (Invitrogen) and affinity purified with Ni-NTA Agarose beads (Qiagen) as previously described [19 (link)]. Briefly, the transformed bacterial cells were first grown in 500 mL batches of Luria-Bertani medium supplemented with 100 μg/mL ampicillin at 37 °C until reaching a 600 nm OD of 0.5. Expression of recombinant Lci2 was induced with 0.2 mM IPTG at 30 °C for 4 h. Bacterial cells were harvested by centrifugation followed by resuspension in 20 mL of denaturing lysis buffer (100 mM sodium phosphate, 10 mM Tris, 8 M Urea, 20 mM imidazole, pH 8.0) and membrane disruption through sonication on ice. For purification, 400 mL of the Ni-NTA beads was added, followed by washes in lysis buffer with 50 mM imidazole (pH 6.0) and elution in the same buffer with 1 M imidazole (pH 4.5), all under denaturing conditions. Purified protein products were analyzed by 15% SDS-PAGE gels stained with Coomassie blue R-250. To determine the recombinant protein concentration, densities of the Lci2 bands in Coomassie blue-stained gels were compared with those of known concentrations of BSA.
5
Recombinant PrP Protein Production
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PJex-WT-PrP plasmid was a generous gift from Dr Glenn Millhauser (the University of California Santa Cruz) and was reengineered to make an S3 equivalent allele using Escherichia coli BL21(DE3)pLysS (Invitrogen). Recombinant proteins have been produced as previously described (110 (link)).
6
Expression and Purification of His-tagged Proteins
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For the expression of His-tagged recombinant proteins, Escherichia coli BL21(DE3) pLysS (Invitrogen) bacteria were transformed with the pRSET derived plasmids, grown in LB medium and expression induced by IPTG. Induced cells were harvested, resuspended in 0.15 M phosphate buffered saline, pH 7.2 (PBS) and lysed by sonication. Protein purification was performed with Ni-NTA Agarose (Qiagen). Protein products were analysed by 15% polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE), followed by staining of the proteins with Coomassie blue R-250. For estimation of the recombinant proteins concentrations, the densities of their stained bands in Coomassie blue stained gels were compared with those of known concentrations of bovine serum albumin (BSA).
7
Expression and Purification of GST-NPP-19
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The expression of NPP-19 full-length N-terminally fused to GST was induced by the addition of 1 mM IPTG to 1 liter of LB cultures of exponentially growing Escherichia coli BL21 DE3 pLysS (Invitrogen) strain [optical density (OD) = 0.6], before incubation for 3 hours at 25°C. After pelleting by centrifugation, the bacteria were resuspended in lysis buffer [0.5 M NaCl, 5% glycerol, 50 mM tris-HCl (pH 8), 1× protease inhibitors (cOmplete Protease Inhibitor Cocktail tablets, Roche)] before lysis by sonication. The soluble portion of the lysate was loaded on 500 μl of glutathione (reduced form) beads (slurry) and incubated for 1 hour. The beads were washed with at least 100 ml of lysis buffer and the proteins were eluted with 20 mM glutathione solution in 50 mM tris-HCl (pH 8). Proteins were aliquoted and flash-frozen in liquid nitrogen and stored at −80°C.
8
Escherichia coli BL21(DE3)pLysS Expression Protocol
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Escherichia coli BL21(DE3)pLysS (Invitrogen) was purchased from Thermo Fisher Scientific Japan. The pRSET‐EmGFP bacterial expression vector was purchased from Thermo Fisher Scientific Japan. The vector pRSET‐EmGFP was introduced to E. coli BL21(DE3)pLysS using the standard method in the users’ manual. The strain was cultivated in LB broth that included 10 g/L Bacto® Tryptone (Becton Dickinson and Co. (BD) Japan), 5 g/L Bacto® yeast extract (BD), and 10 g/L NaCl. The culture was incubated overnight at 37°C with shaking at 200 rpm. This was the inoculum used in the experiments. The culture broth was stored as frozen stocks with 30% glycerol in a deep freezer at −80°C. Experimental‐grade yeast extracts were purchased from BD, Millipore Sigma Japan (Tokyo, Japan), Kyokuto Pharmaceutical Industrial Co. Ltd., and Nihon Pharmaceutical Co. Ltd. (Tokyo, Japan), and referred to as E1, E2, E3, and E4, respectively. Manufacturing‐grade yeast extracts were provided by Oriental Yeast Co. Ltd., and Nihon Paper, and named M1, M2, M3, and M4.
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Escherichia coli BL21(DE3)pLysS (Invitrogen) was purchased from Thermo Fisher Scientific Japan. The pRSET‐EmGFP bacterial expression vector was purchased from Thermo Fisher Scientific Japan. The vector pRSET‐EmGFP was introduced to E. coli BL21(DE3)pLysS using the standard method in the users’ manual. The strain was cultivated in LB broth that included 10 g/L Bacto® Tryptone (Becton Dickinson and Co. (BD) Japan), 5 g/L Bacto® yeast extract (BD), and 10 g/L NaCl. The culture was incubated overnight at 37°C with shaking at 200 rpm. This was the inoculum used in the experiments. The culture broth was stored as frozen stocks with 30% glycerol in a deep freezer at −80°C. Experimental‐grade yeast extracts were purchased from BD, Millipore Sigma Japan (Tokyo, Japan), Kyokuto Pharmaceutical Industrial Co. Ltd., and Nihon Pharmaceutical Co. Ltd. (Tokyo, Japan), and referred to as E1, E2, E3, and E4, respectively. Manufacturing‐grade yeast extracts were provided by Oriental Yeast Co. Ltd., and Nihon Paper, and named M1, M2, M3, and M4.
9
Isolation and Cloning of CsTegu20.6 Gene
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The full-length CsTegu20.6 gene sequence was isolated from an EST library of adult worms using polymerase chain reaction (PCR). C1000 TouchTM Thermal Cycler (Bio-Rad Laboratories Inc., Hercules, CA, USA) and TaKaRa LA Taq polymerase (TAKARA, RR042, Shiga, Japan) were used in the PCR reaction. The forward and reverse primers for CsTegu20.6 were 5′-GGG CAA GGT ACC ATG GAG CCA TTC TTA GAA G-3′ and 5′-CCC GTT AAG CTT TCA GCT TGG TGT CTT CCA C-3', incorporating Kpn I and Hind III restriction sites (underlined), respectively. Cycling conditions were as follows: 95 °C for 30 s, followed by 30 cycles of 94 °C for 30 s, 58 °C for 30 s, 72 °C for 60 s and, finally, 72 °C for 10 min. The amplified PCR products were purified, digested with Kpn I and Hind III endonucleases, run on a 1% agarose gel, excised from the gel and ligated into the bacterial expression vector pRSETb (Invitrogen, Carlsbad, CA, USA). After antibiotic selection, positive clones were confirmed by nucleotide sequencing (Macrogen, Seoul, Korea) and transformed into Escherichia coli BL21 (DE3) pLysS (Invitrogen).
10
Purification of Recombinant Human ApoA-I
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Human ApoA-I proteins, containing at the N-terminus a 6-Histidine-tag and tobacco etch virus (TEV) protease recognition site, were produced in Escherichia coli BL21(DE3) pLysS (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) as described in [42 (link)].
The recombinant proteins were isolated using an immobilized metal affinity chromatography (IMAC, His-Trap-Nickel-chelating columns, GE Healthcare, Chicago, IL, USA) and the his-tag removed by TEV protease treatment. The his-tag free proteins were then purified by performing a second IMAC [43 (link)]. Protein purity was analyzed by SDS-PAGE, followed by Coomassie staining, and protein concentration was determined by using a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific).
The recombinant proteins were isolated using an immobilized metal affinity chromatography (IMAC, His-Trap-Nickel-chelating columns, GE Healthcare, Chicago, IL, USA) and the his-tag removed by TEV protease treatment. The his-tag free proteins were then purified by performing a second IMAC [43 (link)]. Protein purity was analyzed by SDS-PAGE, followed by Coomassie staining, and protein concentration was determined by using a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific).
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