RD1-MART1 and RD1-MART1HIGH were introduced into the pET28a expression vector with a C-terminal AviTag (Avidity) using NcoI and EcoRI restriction sites (forward primer: 5’ - TAT ACC ATG GGC AGC AGC CAT CAT CAT CAT CAT CAC AGC AGC GGC CTG GTG CCG CGC GGC AGC aat gct ggt gta aca caa acg cc - 3’, Reverse primer: 5’ - T TTA GAA TTC TTA ttc gtg cca ttc gat ttt ctg agc ctc gaa gat gtc gtt cag acc gcc acc gtc tgg agt gac cac aac ctg ggt - 3’. Plasmids were transformed into the BL21-DE3 cell line (NEB), expanded, and induced for expression. Following induction, cells were passed through a microfluidizer (Microfluidics Corporation, Newton, MA, USA), inclusion bodies were isolated, and protein was purified as previously described64 (link). Soluble scTCR were refolded and purified with Ni-NTA agarose resin (Qiagen, Valencia, CA) followed by gel filtration (Superdex 200, GE Healthcare). Folded scTCRs were biotinylated in vitro (Avidity, BirA enzyme). Biotinylation was verified by gel-shift with streptavidin by SDS-PAGE.
Bl21 de3 cell line
Manufactured by New England Biolabs
The BL21-DE3 cell line is a widely-used Escherichia coli strain commonly employed for the expression of recombinant proteins. It is designed to facilitate the production of target proteins under the control of the T7 promoter system.
Automatically generated - may contain errors
2 protocols using bl21 de3 cell line
1
Expression and Purification of scTCR Proteins
2
Expression and Purification of scTCR Proteins
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RD1-MART1 and RD1-MART1HIGH were introduced into the pET28a expression vector with a C-terminal AviTag (Avidity) using NcoI and EcoRI restriction sites (forward primer: 5’ - TAT ACC ATG GGC AGC AGC CAT CAT CAT CAT CAT CAC AGC AGC GGC CTG GTG CCG CGC GGC AGC aat gct ggt gta aca caa acg cc - 3’, Reverse primer: 5’ - T TTA GAA TTC TTA ttc gtg cca ttc gat ttt ctg agc ctc gaa gat gtc gtt cag acc gcc acc gtc tgg agt gac cac aac ctg ggt - 3’. Plasmids were transformed into the BL21-DE3 cell line (NEB), expanded, and induced for expression. Following induction, cells were passed through a microfluidizer (Microfluidics Corporation, Newton, MA, USA), inclusion bodies were isolated, and protein was purified as previously described64 (link). Soluble scTCR were refolded and purified with Ni-NTA agarose resin (Qiagen, Valencia, CA) followed by gel filtration (Superdex 200, GE Healthcare). Folded scTCRs were biotinylated in vitro (Avidity, BirA enzyme). Biotinylation was verified by gel-shift with streptavidin by SDS-PAGE.
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