Nebexpress cell free e coli protein synthesis system

In vitro expression of Ehrlichia proteins was performed using the NEBExpress cell-free E. coli protein synthesis system (New England Biolabs, Ipswich, MA). Lyophilized plasmids were reconstituted in water and purified using the UltraClean 96 PCR cleanup kit (Qiagen, Germantown, MD). Plasmids were then added to E. coli extract and a reaction premix in a 96-well plate and incubated at 37°C for 3 h with orbital shaking (300 rpm) according to the manufacturer’s instructions.

The E. coli cell-free expression kit, NEBExpress® Cell-free E. coli Protein Synthesis System, and the restriction enzyme DPNI were bought from New England Biolabs. Chromatographic columns were from Cytiva. Other chemicals were acquired from Sigma-Aldrich.

The pET-22b ( +)-CTX^WT and CTX^VAR plasmids were extracted from glycerol stock of DH10B cells using GF-1 Plasmid DNA Extraction kit (Vivantis, Malaysia) following the manufacturer’s protocol. The CTX^WT and CTX^VAR genes were amplified by touch-down PCR with their respective forward and reverse primers (Supplementary File 1-Table S2).
To express CTX^WT and CTX^VAR, pET-22b ( +) plasmids harboring the respective genes were first transcribed into RNA using the HiScribe™ T7 Quick High Yield RNA Synthesis Kit (New England Biolabs, United States). The transcribed RNAs were then expressed using NEBExpress® Cell-free E. coli Protein Synthesis System (New England Biolabs, United States) for in vitro protein translation, according to the manufacturer's protocol. After that, CTX^WT and CTX^VAR were purified using a HisTrap™ HP, 1 mL affinity column on ÄKTA Pure protein purification system (Cytiva, United States). The HisTrap column was equilibrated with buffer A (20 mM sodium phosphate, 0.5 M NaCl, pH 8.0) before the samples were injected into the column. CTX^WT and CTX^VAR were purified with a gradient elution from 4 to 100% of buffer B (20 mM sodium phosphate, 0.5 M NaCl, 500 mM imidazole, pH 8.0) at a flow rate of 500 uL/min. The presence of his-tagged expressed CTX and its purity were visualized by western blot.