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We set out to develop an easy-to-use system for in vitro synthesis of recombinant eukaryotic proteins containing posttranslational modifications. A wide range of eukaryotic proteins require such modifications (e.g., phosphorylation, glycosylation, signal peptide cleavage) for correct folding or to display full functional activity.
To optimize expression, a number of expression constructs were generated using polymerase chain reaction (PCR). These constructs encoded proteins carrying combinations of C- or N-terminal His- or Strep-affinity tags. Promoters added during the PCR enabled the efficiency of expression and proportion of soluble protein delivered by each construct to be tested directly in small-scale synthesis reactions. Once the makeup of the optimal construct was determined, it was cloned into an expression plasmid for larger-scale syntheses. A range of proteins—including kinases, membrane proteins, and transcription factors— were expressed to test the robustness and applicability of the method. Optimized Spodoptera frugiperda cell lysates, which contain all the cellular machinery required for posttranslational modifications, were used for expression. The presence of post-translational modifications in synthesized proteins was demonstrated using epitope-specific antibodies (phosphoproteins), enzymatic treatment (glycoproteins), and activity assays (luciferase).
The ability to use PCR products in small-scale expression screening reactions speeded up the entire process of construct optimization considerably. It was possible to synthesize a wide range of post-translationally modified proteins in high yields (up to 40 μg/mL reaction), significantly higher than those obtained in a comparison with a rabbit-reticulocyte lysate-based system.
This system offers a range of benefits for eukaryotic protein expression, including simplified expression screening, a fast procedure, robust and high-yield expression, and no requirement for specialized equipment.