gBlocks for the chimeric proteins hCLDN1 (accession number O95832), hCLDN2 (accession number O15551), hCLDN3 (accession number O15551), hOCLN (accession number Q16625), enterotoxin Clostridium perfringens (CPE) (accession number CAA57443), mutant CPE(m19) [68 (link)] and E-CAD (Val102-Asp312, accession number P12830) were obtained from IDT DNA Technologies https://www.idtdna.com/pages (Supplementary Information) (accessed on 5 March 2020), codon optimized for E. coli K-12 (IDT DNA Technologies Codon Optimization Tool). The gBlocks were amplified with forward and reverse primers, adapters of T7 promoter primer and T7 reverse primer, followed by restriction enzyme digestion (XhoI and NdeI). Fragments were subcloned in pET28a-MBP plasmid, kanamycin resistant (Supplementary Information). The final product produces an N-terminal MBP-fusion protein of the target with a C-terminal 6xHis tag. Cloning and subcloning transformations performed in NEB 5-alpha (NEB). Plasmids for protein expression were transformed into SHuffle T7 Express (NEB), spectinomycin resistant. Protein expression and purification (Amylose resin) were performed following manufacturer’s instructions. Eluate was concentrated by using Microsep Advance with 10k Omega centrifugal devices from Pall Corporation https://www.pall.com/ (accessed on 5 March 2020).
Shuffle t7 express
Manufactured by New England Biolabs
Sourced in Germany, United States
The SHuffle T7 Express is a competent E. coli strain designed for the expression of disulfide-bonded proteins. It contains the SHuffle T7 Express plasmid, which carries the T7 RNA polymerase gene under the control of the lacUV5 promoter, allowing for high-level expression of target proteins.
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Lab products found in correlation
Fetal calf serum (fcs), by Nichirei Biosciences (2 mentions)
Pyridylaminated pa sugars, by Takara Bio (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Rnalater, by Thermo Fisher Scientific (2 mentions)
Pgem t easy, by Promega (2 mentions)
Pet28a, by Merck Group (2 mentions)
Neb 5 alpha, by New England Biolabs (1 mentions)
Microsep advance, by Pall Corporation (1 mentions)
Novablue, by Merck Group (1 mentions)
Expi293f cells, by Thermo Fisher Scientific (1 mentions)
Expifectamine 293 transfection kit, by Thermo Fisher Scientific (1 mentions)
Hitrap protein a hp column, by GE Healthcare (1 mentions)
Hitrap desalting column, by GE Healthcare (1 mentions)
Kta pure chromatography system, by GE Healthcare (1 mentions)
Hiload superdex 75 pg column, by GE Healthcare (1 mentions)
Vibra cell vcx500, by Sonics (1 mentions)
E coli bl21, by New England Biolabs (1 mentions)
Bl21 gold de3, by Agilent Technologies (1 mentions)
Bl21 de3, by New England Biolabs (1 mentions)
Wild type female c57bl 6 mice, by Jackson ImmunoResearch (1 mentions)
17 protocols using shuffle t7 express
1
Expression and Purification of Recombinant Fusion Proteins
2
Chimera 1 Protein Expression
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The synthetic gene of chimera 1 (Qm1) GenScript (Nanjing, China) was cloned into XhoI-NdeI sites of the vector pET-22b (+). E. coli strain SHuffle® T7 Express (New England Biolabs, Ipswich, MA, USA) was used as the expression strain.
3
Recombinant protein production protocols
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The Escherichia coli strains NovaBlue (Novagen, Merck Millipore, Germany) and SHuffle®T7 Express (C3028H, New England Biolabs, Germany) were routinely grown as described previously [6 (link)]. Transformation and selection were carried out following protocols already published [6 (link)]. Experiments to produce WbgU were carried out using TB Medium supplemented with 0.5% d -glucose (Glc). For the production of unglycosylated T7Muc10 5010 Medium (50 g/L yeast extract, 10 g/L peptone, 0.492 g/L magnesium sulfate heptahydrate) was used. Glycosylated T7Muc10 was expressed as described previously in a 24-deepwell plate containing 2 mL EnPresso® B medium in each well and pooled for purification [6 (link)].
4
Quantitative Assessment of E. coli Periplasmic Protein
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Bacterial cultures of E. coli Shuffle T7 Express (New England BioLabs) cells carrying either a control plasmid or a periplasmic tpeV construct were grown overnight in liquid LB medium supplemented with 0.2% glucose and ampicillin at 37°C with shaking. Cells were washed three times with LB and 100× back-dilutions were made in fresh liquid LB medium with 500 μM IPTG (or 0.2% glucose for uninduced controls) and ampicillin. Strains were incubated at 37°C for 2 h, washed three times with phosphate-buffered saline (PBS), and normalized to an OD600 of 1. One hundred microliters of each culture was incubated with 1 μl propidium iodide (1 mg/ml) for 15 to 30 min. Fluorescence values were taken on a Synergy BioTek plate reader using an excitation ƛ of 535 nm and emission ƛ of 617 nm and normalized by subtracting the average values from samples with propidium iodide but no cells. Data represent the averages obtained from seven biological replicates from two independent experiments.
5
Recombinant scFv and Fab-Fc Production
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Reformatting, expression and purification of scFvs was performed as described previously (89 (link)). Briefly, isolated yeast vectors were sequenced and scFv encoding genes were reformatted into a pET30 plasmid using golden gate assembly, followed by recombinant expression in E. coli SHuffle® T7 Express (New England Biolabs). A two-step affinity purification was performed including IMAC and Strep-Tactin®XT purification, followed by buffer exchange against PBS. Production of Fc-fused scFvs and full-length antibodies (Fab-Fc) was conducted with pTT5-derived golden gate assembly vectors in Expi293F™ cells (Thermo Fisher Scientific). Expi293F™ cells were transiently transfected using ExpiFectamine™ 293 Transfection Kit (Thermo Fisher Scientific) following the manufacturer's protocol. For purification of Fc-containing antibody constructs, cell culture supernatants were collected five days post transfection, sterile filtered and applied to a HiTrap™ Protein A HP column (GE Healthcare) using an ÄKTA pure™ chromatography system (GE Healthcare). Buffer exchange against PBS or TBS was performed using a HiTrap™ Desalting column (GE Healthcare).
6
Purification and characterization of marine lectins
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K. alvarezii lectins KAA-1 and KAA-2 (previously declared as ECA-1 and ECA-2) and Eucheuma serra lectin ESA-2 were prepared as described previously (Kawakubo et al. 1997 (link), 1999 (link)) and kept at −20 °C until use. The cultivated specimen of K. alvarezii was stored in RNAlater (Life Technologies, CA, USA) at −20 °C until RNA extraction. A vector pGEM-T Easy (Promega, WI, USA) and E. coli strain DH-5α were used for subcloning PCR amplified products. An expression vector pET-28a(+) (Merck, Darmstadt, Germany) and an E. coli strain SHuffle T7 Express (New England Biolabs, MA, USA) were used for recombinant lectin preparation. Pyridylaminated (PA)-sugars were purchased from Takara Bio (Tokyo, Japan), except PA-derivatives of oligomannosides including mannotriose (Manα1-6(Manα1-3)Man-OH) and mannopentaose (Manα1-6(Manα1-3)Manα1-6(Manα1-3)Man-OH) which were previously prepared (Hori et al. 2007 (link)). A recombinant glycosylated HIV-1 IIIB gp120 (baculovirus) was purchased from ImmunoDiagnostics (MA, USA). Jurkat cells and T lymphocyte-derived cells were propagated in RPMI 1640 medium (Life Technologies) supplemented with 10 % fetal calf serum (Nichirei Biosciences, Tokyo, Japan).
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K. alvarezii lectins KAA-1 and KAA-2 (previously declared as ECA-1 and ECA-2) and Eucheuma serra lectin ESA-2 were prepared as described previously (Kawakubo et al. 1997 (link), 1999 (link)) and kept at −20 °C until use. The cultivated specimen of K. alvarezii was stored in RNAlater (Life Technologies, CA, USA) at −20 °C until RNA extraction. A vector pGEM-T Easy (Promega, WI, USA) and E. coli strain DH-5α were used for subcloning PCR amplified products. An expression vector pET-28a(+) (Merck, Darmstadt, Germany) and an E. coli strain SHuffle T7 Express (New England Biolabs, MA, USA) were used for recombinant lectin preparation. Pyridylaminated (PA)-sugars were purchased from Takara Bio (Tokyo, Japan), except PA-derivatives of oligomannosides including mannotriose (Manα1-6(Manα1-3)Man-OH) and mannopentaose (Manα1-6(Manα1-3)Manα1-6(Manα1-3)Man-OH) which were previously prepared (Hori et al. 2007 (link)). A recombinant glycosylated HIV-1 IIIB gp120 (baculovirus) was purchased from ImmunoDiagnostics (MA, USA). Jurkat cells and T lymphocyte-derived cells were propagated in RPMI 1640 medium (Life Technologies) supplemented with 10 % fetal calf serum (Nichirei Biosciences, Tokyo, Japan).
7
Recombinant Anti-HER2 scFv Expression
8
Recombinant Expression and Purification of Sc Wsc Proteins
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CRDs of ScWsc1, ScWsc2, and ScWsc3 were produced following a low temperature protocol [38 (link)] using the expression plasmids BHUM3120, BHUM3121, and BUM3122, and the E. coli strain SHuffle T7 express (New England Biolabs GmbH, Frankfurt, Germany). Proteins were purified by Ni-NTA affinity chromatography (Macherey-Nagel, Düren, Germany) and subsequent size exclusion chromatography using a HiLoad Superdex 75 pg column (GE Healthcare, Munich, Germany). All steps were carried out in AML buffer (20 mM Tris/HCl, pH 8.0, 350 mM NaCl).
9
Bacterial Strains for Protein Expression
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E. coli strain DH5α was used for plasmid construction while SHuffle T7 Express (New England Biolabs)20 (link) was used for cyclonal expression and library selections. Protein antigens for immunoassays including GST-Gcn4-PP, GST-HAG, GST-c-Myc, and GST-c-Jun were expressed using E. coli T7 Express (New England Biolabs).
10
Recombinant anti-VT2 mAb production
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To produce the Zipbody with an SKIK tag, Hc and Lc were coexpressed in E. coli Shuffle T7 Express (New England Biolabs, Ipswich, MA, USA) as the host, grown in 50 mL LB medium supplemented with 100 µg/mL ampicillin with 1 mM IPTG induction at 16 °C for 24 h, as described in our previous study [17 (link)]. The inclusion bodies were refolded by the stepwise dialysis method using guanidinium hydrochloride [20 (link)]. In the case of anti-nontoxic VT2 mAb clone, Hc and Lc genes independently cloned into pET22b were separately expressed in E. coli BL21star (DE3) hovering plasmid pSJS1240 in the same medium with 100 µg/mL spectinomycin at 37 °C for 4.5 h. The amount of inclusion bodies of Hc and Lc was equalized and refolded together in the single dialysis tube. The KD values were measured by Bio-layer interferometry method.
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