Traditional cell-based methods of expressing proteins are encumbered by the necessity to maintain viability of the cell line requiring tight control over multiple biochemical and biophysical parameters. Requirement of the intact cell wall also prevents the efficient incorporation of non-native amino acids (nnAA) due to the lack of endogenous transport mechanisms in these systems. The XpressCF platform overcomes these significant limitations by separating the precise cellular machinery required for transcription, translation, and energy production into an E.coli-derived mixture, or Extract capable of continuous oxidative phosphorylation. The addition of DNA plasmid, coding for nnAA if applicable, and polymerase to the extract triggers protein production and is sustained with glucose, an inexpensive energy source. This cell-free system is manipulated to facilitate optimal protein folding by altering pH, temperature, redox potential, ionic strength, etc. to identify an optimal set of conditions to produce antigenic proteins in the desired conformation. Proteins can be rapidly engineered and optimized by producing multiple variants in parallel from DNA libraries.
Industrialized & Scaled
The primary invention underpinning the XpressCF platform was the creation of engineered E.coli-derived proprietary extracts containing fully functional oxidative phosphorylation machinery in inverted membrane vesicles and orthogonal tRNA capable of catalyzing nnAA incorporation. This innovation enables the Extract to regenerate high energy phosphates required for protein translation and nnAA incorporation, reducing production cost and permitting the use of industrial sized reaction vessels and longer manufacturing run times than previously thought possible. The XpressCF system routinely produces g/L of complex proteins in 8-10 hours and scales linearly from bench to large-scale GMP production. The protein of interest is produced at high yield and the absence of cellular debris combine to simplify downstream purification, further enhancing production efficiency.