Recombinant overexpression of SidJ99-C and full-length Calmodulin were achieved through expression using E. coli OneShot BL21 Star (DE3) cells (Invitrogen). The cells were cultivated at 37°C in lysogeny broth supplemented with 25 µg/mL Kanamycin and 34 µg/mL Chloramphenicol for SidJ99-C and 100 µg/mL Ampicillin and 34 µg/mL Chloramphenicol for Calmodulin. Both SidJ99-C and Calmodulin were expressed, lysed, and purified using the same protocol as the SidJ/CaM complex described in the previous section, but Calmodulin was incubated in lysis buffer with 4mM EDTA added at 4°C for 2 hours before size exclusion chromatography, and neither protein were cleaved or dialyzed.
One shot bl21 star de3 cells
Manufactured by Thermo Fisher Scientific
Sourced in Denmark
One Shot® BL21 Star™ (DE3) cells are a specialized strain of Escherichia coli (E. coli) cells designed for the expression of recombinant proteins. These cells provide efficient protein expression under the control of the T7 RNA polymerase promoter system.
Automatically generated - may contain errors
5 protocols using one shot bl21 star de3 cells
1
Recombinant Expression of SidJ99-C and Calmodulin
2
Recombinant Expression of SidJ99-C and Calmodulin
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Recombinant overexpression of SidJ99-C and full-length Calmodulin were achieved through expression using E. coli OneShot BL21 Star (DE3) cells (Invitrogen). The cells were cultivated at 37°C in lysogeny broth supplemented with 25 µg/mL Kanamycin and 34 µg/mL Chloramphenicol for SidJ99-C and 100 µg/mL Ampicillin and 34 µg/mL Chloramphenicol for Calmodulin. Both SidJ99-C and Calmodulin were expressed, lysed, and purified using the same protocol as the SidJ/CaM complex described in the previous section, but Calmodulin was incubated in lysis buffer with 4mM EDTA added at 4°C for 2 hours before size exclusion chromatography, and neither protein were cleaved or dialyzed.
3
Purification and Cysteine Modification of Recombinant PPARδ
4
Recombinant SpyCatcher-AP205-L2 CLP Vaccine
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SpyCatcher-AP205-L2 was designed, recombinantly expressed, and purified as described in [19 (link)]. Briefly, the RG1 epitope of the minor capsid protein (L2) of Human Papilloma Virus (HPV) 16 (QLYKTCKQAGTCPPDIIPKVEG) was attached by PCR amplification to the 3′ end of the SpyCatcher-AP205 construct [14 (link)]. The SpyCatcher-AP205-L2 construct was inserted into a pet-15b vector, expressed in One Shot® BL21 Star™ (DE3) cells (Thermo Scientific), and purified on Optiprep™ (Sigma-Aldrich, Denmark) step gradients, as previously described [14 (link),19 (link),22 (link)]. Throughout this article, the above described SpyCatcher-AP205-L2 CLP vaccine is referred to as the “CLP vaccine”.
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.
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