Final constructs were co-transformed into OneShot BL21 StarDE3 (Life technologies). Protein expression was induced as previously described, using IPTG and chloramphenicol [22 (link)]. Fluorescence was measured either by flow cytometry or using a fluorescent plate reader using appropriate filters and excitation wavelengths. The reported values represent means obtained from three independent experiments carried out on separate days that were expressed after subtraction of background fluorescence (cells expressing only N- and C- terminal GFP fragments, without potential interaction partners). Binding of C-terminal fragments of PfHsp70 and PfHsp90 to TPR motifs of PfHop was reported relative to the highest measured fluorescence and is thus referred to as "relative binding". P-values to validate the relative affinities of PfHop TRP domains for C-terminal fragments of PfHsp70-1 and PfHsp90 were calculated using the student’s t-test.
One shot bl21 star de3
Manufactured by Thermo Fisher Scientific
The One Shot BL21 Star (DE3) is a competent Escherichia coli (E. coli) strain designed for high-efficiency protein expression. It is optimized for the expression of recombinant proteins under the control of the T7 promoter system.
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Lab products found in correlation
Glutathione sepharose 4b, by GE Healthcare (2 mentions)
S adenosyl l methyl 3h methionine, by PerkinElmer (1 mentions)
Ni nta agarose bead, by Qiagen (1 mentions)
Pgex 4t 1 vector, by GE Healthcare (1 mentions)
Amersham hyperfilm mp, by GE Healthcare (1 mentions)
En3hance, by PerkinElmer (1 mentions)
Cos 7, by American Type Culture Collection (1 mentions)
Hek293, by American Type Culture Collection (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Pgex 4t 1 expression vector, by GE Healthcare (1 mentions)
One shot bl21 star de3 cells, by Thermo Fisher Scientific (1 mentions)
Hiload superdex 75pg, by GE Healthcare (1 mentions)
H2030, by American Type Culture Collection (1 mentions)
One shot top10, by Thermo Fisher Scientific (1 mentions)
Calu 1, by American Type Culture Collection (1 mentions)
Mia paca 2, by American Type Culture Collection (1 mentions)
Hct116, by American Type Culture Collection (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
6 protocols using one shot bl21 star de3
1
Fluorescence-based Protein-Protein Interaction Assay
2
In Vitro Methylation Assay for Protein Kinase
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The in vitro methylation assay was performed according to methods previously described in [36 (link)]. The coding sequence of KIN was cloned in pET-23a(+) vector (EMD Chemicals, Burlington, VT, USA), and methyltransferases were cloned in to pGEX-4-T1 vector (GE Healthcare, Chicago, IL, USA). Vectors were transformed in One Shot BL21 Star (DE3) (Life Technologies) and protein synthesis was induced with IPTG. Bacteria were harvested by centrifugation and pellets were lysed with the use of an IEC French Press (Thermo Scientific). The resulting proteins were purified using Ni-NTA Agarose beads (QIAGEN, Hilden, Germany), and Glutathione Sepharose 4B (GE Healthcare, Chicago, IL, USA), according to the manufacture’s recommendations. For each reaction, 1 μg of GST-tagged methyltransferase was incubated with 2.5 μg His-tagged KIN and 5 μCi of S-(methyl-3H)-Adenosyl-L-methionine (81.7 Ci/mmol; PerkinElmer, Norwalk, CT, USA), according to the manufacture’s specifications. The samples were resolved in a 10% acrylamide gel which was then treated with EN3HANCE (PerkinElmer), according to the manufacturer’s recommendations. Tritium-based methylation signals were detected by radiography after 24 h of exposure on Amersham Hyperfilm MP (GE Healthcare, Chicago, IL, USA) at −80 °C.
3
Cfap418 Knockout Mouse Model
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Cfap418–/– mice with a mixed CBA/C57BL6 genetic background have been described previously (15 (link)). Mice of both sexes were randomly assigned to experimental groups at various time points. Littermate Cfap418+/+ or Cfap418+/– mice were included as controls. HEK293 (CRL-10852) and COS-7 (CRL-1651) cell lines were purchased from ATCC (https://www.atcc.org ). These cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum, 50 U/mL penicillin, and 50 mg/mL streptomycin (Thermo Fisher Scientific). The One Shot BL21 Star (DE3) cell line was purchased from Thermo Fisher Scientific (C601003) and cultured according to the manufacturer’s protocol.
4
Recombinant TbLysoPLA Antibody Production
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A recombinant T. brucei full length LysoPLA fused to a GST tag on its N-terminal extremity, was expressed in E. coli One Shot BL21star (DE3) (Thermofisher) using the pGEX4T1 expression vector (GE Healthcare). Protein expression was induced at 37 °C for 3 h using 0.5 mM isopropyl-d -thiogalactopyranoside. The cells were harvested by centrifugation, resuspended in PBS and sonicated. Proteins released in the soluble form were purified using Glutathione Sepharose 4B according to the manufacturer’s instructions (GE Healthcare). On-column thrombin digestion was performed to release the protein without the GST tag, then dialysed against PBS. Purified recombinant TbLysoPLA was used as an antigen to raise polyclonal antibodies. Two rabbits were injected 4 times at 15-days intervals using Covalab facilities (www.covalab.com ).
5
Production and Purification of SpyCatcher and HPV Antigens
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To produce the recombinant SpyCatcher control antigen, a flexible Glycine-Glycine-Serine (GGS) linker, followed by a hexahistidine (6xHis)-tag was added to the C-terminus of SpyCatcher. SpyCatcher was produced in One Shot® BL21 Star™ (DE3) cells (Thermo Scientific) and purified using ion metal affinity chromatography as well as ion exchange chromatography. This protein was used as the coat for anti-SpyCatcher IgG ELISA and as the control antigen in mouse immunizations (described below)
The RG1 epitope of HPV16 (QLYKTCKQAGTCPPDIIPKVEG) was attached by overlap extension PCR to the 5′ end of a biologically irrelevant carrier protein (accession number WP_057363222, amino acid 44-338). The HPV peptide/protein fusion was expressed in One Shot® BL21 Star™ (DE3) (Thermo Scientific) and purified using ion metal affinity chromatography as well as size exclusion chromatography (HiLoad Superdex 75pg, GE Healthcare). This protein was used for coat protein in the anti-HPV peptide ELISAs.
The RG1 epitope of HPV16 (QLYKTCKQAGTCPPDIIPKVEG) was attached by overlap extension PCR to the 5′ end of a biologically irrelevant carrier protein (accession number WP_057363222, amino acid 44-338). The HPV peptide/protein fusion was expressed in One Shot® BL21 Star™ (DE3) (Thermo Scientific) and purified using ion metal affinity chromatography as well as size exclusion chromatography (HiLoad Superdex 75pg, GE Healthcare). This protein was used for coat protein in the anti-HPV peptide ELISAs.
6
Cell Line and Bacterial Strain Acquisition
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Cell lines and bacterial strains H358, MIA-PaCa2, SW837, CALU1, H23, H2122, SW1463, H2030, H1373, H1792, SW1573, A549, H441, HCT116, and A375 cells were purchased from the American Type Culture Collection (ATCC). LU65 (JCRB0079) and LU99A (JCRB0044) were purchased from the Japanese Collection of Research Bioresources Cell Bank (JCRB). HCC44 (ACC 534) was purchased from DSMZ. HCC1171 (71171) was purchased from the Korean Cell Line Bank (JCLB). Ras-less MEFs were obtained from the NIH (NCI RAS initiative at the FNLCR). All cancer cell lines at Wellspring Biosciences underwent identity validation by SNP genotyping authentication services and were tested for mycoplasma infection (IDEXX Bioresearch) before studies were conducted. All in-house cell lines within the embodied work were carried for no longer than 20 cell passages. Cells were maintained in a humidified incubator at 37 C with 5% CO 2 , and grown in RPMI 1640 or DMEM supplemented with 10% FBS (GIBCO) and 50 IU ml -1 penicillin/streptomycin (GIBCO). E. Coli cells for cloning (One Shot TOP10) and protein production (One Shot BL21 Star (DE3)) were purchased from ThermoFisher Scientific. For custom heavy isotopically labeled KRAS protein standards produced at PROMISE Advanced Proteomics (Grenoble, France) the E. Coli strain BL21(DE3) auxotrophic for lysine and arginine (genotype: lysA -, argA -) was used.
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