AAT Bioquest

What are the types of extracts used for cell-free protein expression?

Posted August 5, 2023


Extracts used for cell-free protein expression typically are: E. coli, insect cells, wheat germ, or rabbit reticulocytes. The E. coli extract-based CF expression system is the most commonly used because they produce a very high protein yield, are relatively tolerant of additives, and are cost effective. Advances in the scientific field led to the production of eukaryotic in vitro translation systems from rabbit reticulocytes, insect cell lysates (e.g. SF9 or SF21) and wheat germ extracts. Extracts made from these systems are composed of the necessary molecules (ribosomes, translation factors, and tRNAs) required for protein synthesis. Lysates from E. coli and wheat germ do not contain endogenous genetic messages. In contrast, lysates made from insect cells and rabbit reticulocytes contain endogenous mRNAs which are translated during synthesis. The advantages of using rabbit reticulocytes include it being part of the mammalian system and it uses cap independent translation. Wheat germs have been optimized over the years by applying additives to ensure correct disulfide bonding or to include isotopic labels. Therefore, the system has been shown to be effective for generating correctly folded or labeled products. Wheat germs may also translate large proteins. Insect cells have the advantage of being capable of protein glycosylation, and they contain endogenous mammalian proteins. 

Additional resources

Optimising protein synthesis in cell‐free systems, a review

Antibodies and Proteomics