FIG 1.

Schematic representation of the expression of nnAA-containing IpaB using XpressCF⁺ CFPS technology. Cell-free protein synthesis (CFPS) for expression of nnAA (pAMF) containing IpaB was performed by generating a reaction mixture comprising (1) Escherichia coli cell extract, (2) components necessary for transcription (T7 polymerase and the DNA template), and (3) molecular machinery facilitating site-selective pAMF incorporation (synthetase, orthogonal tRNA [otRNA], and pAMF) along with (4) essential components necessary for energy production (nucleoside triphosphates [NTPs], amino acids, salts, and cofactors). Postexpression, CFPS product was harvested, and target protein (IpaB-var) was purified and analyzed by SDS-PAGE analysis. pAMF incorporation was confirmed through labeling with DBCO-TAMRA dye. var, variant.