Cell-Free Systems Based on CHO Cell Lysates: Optimization Strategies, Synthesis of “Difficult-to-Express” Proteins and Future Perspectives

Abstract

Nowadays, biotechnological processes play a pivotal role in target protein production. In this context, Chinese Hamster Ovary (CHO) cells are one of the most prominent cell lines for the expression of recombinant proteins and revealed as a safe host for nearly 40 years. Nevertheless, the major bottleneck of common in vivo protein expression platforms becomes obvious when looking at the production of so called “difficult-to-express” proteins. This class of proteins comprises in particular several ion channels and multipass membrane proteins as well as cytotoxic proteins. To enhance the production of “difficult-to-express” proteins, alternative technologies were developed, mainly based on translationally active cell lysates. These so called “cell-free” protein synthesis systems enable an efficient production of different classes of proteins. Eukaryotic cell-free systems harboring endogenous microsomal structures for the synthesis of functional membrane proteins and posttranslationally modified proteins are of particular interest for future applications. Therefore, we present current developments in cell-free protein synthesis based on translationally active CHO cell extracts, underlining the high potential of this platform. We present novel results highlighting the optimization of protein yields, the synthesis of various “difficult-to-express” proteins and the cotranslational incorporation of non-standard amino acids, which was exemplarily demonstrated by residue specific labeling of the glycoprotein Erythropoietin and the multimeric membrane protein KCSA.

Cell-Free Protein Synthesis: A Promising Option for Future Drug Development

“…For complex proteins, eukaryotic CF systems are ideal as they contain the endogenous microsomes derived from the endoplasmic reticulum (ER), enabling co-translational translocation of proteins and ER-based PTMs [10,11,18,19]. There has been a constant improvement in the quality of lysate preparation, system optimization, linear templatebased protein synthesis, and reduction of process costs, which has led to the preparation of cost-effective systems suitable for commercial purposes.…”

“…Toxic proteins may be difficult to synthesize [16] Membrane proteins Suitable for a wide range of MPs of different sizes MP overexpression can lead to cell toxicity and death [17][18][19] Membrane protein solubilization To solubilize MPs, supplements can be added directly to the reaction mixture in the form of nanodiscs, detergents or liposomes (prokaryotic systems and wheat germ systems) or by using endogenous microsomes or proteoliposomes (eukaryotic sys-…”

“…However, these cells have limitations when it comes to difficult-to-express proteins like overexpression of complex MPs, toxic proteins, and multi-subunit proteins as discussed above. CF systems based on CHO lysates are evolving as an alternative strategy for the expression of difficult-to-express proteins [13,[17][18][19]46]. Apart from many general advantages of CF systems, CHO-based CF systems retain most of the features of CHO cells while being more flexible due to the lack of cell membrane boundaries.…”

Comparison of cell‐based versus cell‐free mammalian systems for the production of a recombinant human bone morphogenic growth factor

“…CHO cell lysates were chosen, since they had previously been established as the most productive eukaryotic system in our hands [24], while concomitantly enabling a direct comparison to the production of hBMP2 in cell line 4D6. Previous observations have shown that adding specific circular DNA templates (plasmids) to CHO cell-free protein synthesis systems instead of linear DNA, may further increase protein yields [49]. When added to the cell-free reaction mixture, the PCR product (for details see Fig.…”

“…This corresponds to a 20-fold increase in protein yield compared to the best results from the cell-based expression systems. Previous observations have shown that adding specific circular DNA templates (plasmids) to CHO cell-free protein synthesis systems instead of linear DNA, may further increase protein yields [49]. To exclude any background translational activity of the lysates, a blank control, lacking the supplemented DNA, was prepared.…”