A cell-free enzymatic activity assay for the evaluation of HIV-1 drug resistance to protease inhibitors
Current cell-free protein synthesis systems can synthesize proteins with high speed and accuracy, but produce only a low yield because of their instability over time. Here we describe the preparation of a highly efficient but also robust cell-free system from wheat embryos. We first investigated the source of the instability of existing systems in light of endogenous ribosome-inactivating proteins and found that ribosome inactivation by tritin occurs already during extract preparation and continues during incubation for protein synthesis. Therefore, we prepared our system from extensively washed embryos that are devoid of contamination by endosperm, the source of tritin and possibly other inhibitors. In a batch system, we observed continuous translation for 4 h, and sucrose density gradient analysis showed formation of large polysomes, indicating high protein synthesis activity. When the reaction was performed in a dialysis bag, enabling the continuous supply of substrates together with the continuous removal of small byproducts, translation proceeded for >60 h, yielding 1-4 mg of enzymatically active proteins, and 0.6 mg of a 126-kDa tobacco mosaic virus protein, per milliliter of reaction volume. Our results demonstrate that plants contain endogenous inhibitors of translation and that after their elimination the translational apparatus is very stable. This contrasts with the common belief that cell-free translation systems are inherently unstable, even fragile. Our method is useful for the preparation of large amounts of active protein as well as for the study of protein synthesis itself.