Co-expression with the Type 3 Secretion Chaperone CesT from Enterohemorrhagic E. coli Increases Accumulation of Recombinant Tir in Plant Chloroplasts
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| Peptide Synthesis |
Liquid protein samples were combined with 5x loading buffer
(0.3 M Tris-HCl pH 8.0, 5% SDS, 10% glycerol, 100 mM DTT,
0.05% Phenol Red), heated at 99◦C for 5 min, then loaded onto
Express Plus PAGE 4-20% gels (Genscript Inc., Piscataway, NJ,
USA). Gels were run at 100 V for 100–110 min, then transferred
to polyvinylidine difluoride (PVDF) membrane using the TransBlot
Turbo transfer system (Bio-Rad Laboratories Inc., Hercules,
CA, USA). Blots were blocked with 5% skimmed milk in trisbuffered
saline, pH 7.5, and proteins of interest were probed with
anti-c-myc antibody from mouse (Genscript Inc., Piscataway, NJ,
USA) and the One-Hour Basic Western kit for mouse primary
antibody (Genscript Inc., Piscataway, NJ, USA).Protein sizes were predicted
based on amino acid sequence only, using an online peptide
calculator tool (Genscript Inc.; https://www.genscript.com/sslbin/peptide_mw). |
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chloroplast targeting, EHEC, EPEC, fluorescence microscopy, T3SS, translocated intimin receptor, transplastomic, subunit vaccine
