Mythbusting #4 : “Cell-free systems only use bacterial extract.”

Posted by Bruno Tillier on 25-Apr-2019 14:03:00
Bruno Tillier

bacterial protein (1)

Escherichia coli (E. Coli) is the most popular cell extract source for cell-free expression systems. However, extracts from wheat germ, rabbit reticulocytes or insect cells are also commonly used. Other mammalian cell lines are also more recently used for cell-free protein biosynthesis. Since they present different properties and advantages, the source of cell extract will be selected according to the properties of the protein to synthesize, such as the need for post-translational modifications, and other constraints, like yield, costs or convenience of cell extract preparation (Carlson et al., 2012; Casteleijn et al., 2013, Zemella et al., 2015).

 

Cell-free systems use bacterial extract but also eukaryotic ones according to the protein to synthesize, allowing the best choice for your projects !

Firstly, the origin and biochemical characteristics of the protein to synthesize is decisive in the choice of the cell extract source (Table 1). For example, proteins containing disulfide bonds can be expressed in all systems, except in rabbit reticulocytes. In contrast, proteins requiring post-translational modifications such as glycosylation have to be expressed in cell extracts from rabbit reticulocytes, insect cells or CHO cells.

Table 1. Comparison of different cell-free systems

 

E. coli

Rabbit reticulocytes lysate

Wheat germ

Insect cells

Mammalian cells

Disulfide bonds

Yes

Yes

Yes

Yes

Yes

Post-translational modifications

No

Possible in the presence of microsomal membranes

Possible in the presence of microsomal membranes

Yes

Yes

Membrane proteins expression

Yes

Yes

Yes

Yes

Yes

Typical yield range

1 mg ml-1  to 100 mg ml-1

<100 µg ml -1

 

Up to 10 mg ml -1

< 100 µg ml -1

Up to 750 µg ml -1

 

Although the potential to produce complex proteins is increased using eukaryotic cell-free systems, Escherichia coli is the cell type of bacteria which presents many advantages as a source of cell extract. The preparation of this cell extract is :

  • simple,
  • robust and
  • inexpensive.

In addition, cell extracts from E. coli and wheat germ achieve the highest protein yields, well above those obtained with other eukaryotic cell-based systems.

As an illustration, the production of full-length and smaller formats of antibodies exploits the advantages of either prokaryotic or eukaryotic cell-free expression systems (Stech et al., 2015).
High production yields have been obtained using prokaryotic systems in the presence of PDI or DsbC and variable ratios of GSSG and GSH: up to 950 mg/L for a scFv, 300 mg/L for a Fab and 400 mg/L of an aglycosylated IgG were obtained (Yin et al. 2012).
In addition, the production of functional scFv antibody fragments has been achieved by using vesicle-containing Spodoptera frugiperda (Sf21) insect cell-free expression systems but with yields of 10-15 µg/mL approximately (Stech et al., 2014).

In summary, the cell extract source has an important impact on protein biosynthesis, in particular for the expression feasibility, yield and cost. Thus, the protein properties and production constraints should be carefully analyzed ahead of the selection of the cell extract source to be tested in cell-free expression system experiments. Yhen you have a panel of choices to get to the best results for your protein production project. 

prokaryoticDifferent examples of efficient extracts used with cell-free expression systems

 

mythbustings about cell free technology 

Authors & sources :

Carlson E.D., Gan R., Hodgman C.E., Jewett M.C. 2012. Cell-free protein synthesis: Applications come of age. Biotechnology Advances 30:1185–1194.

Casteleijn M.G., Urtti A., Sarkhel S. 2013. Expression without boundaries: cell-free protein synthesis in pharmaceutical research. International Journal of Pharmaceutics 440:39-47.

Stech M., Hust M., Schulze C., Dübel S., Kubick S. 2014. Cell-free eukaryotic systems for the production, engineering and modification of scFv antibody fragments. Engineering in Life Sciences. 14:387–398.

Stech M., Kubick S. 2015. Cell-Free Synthesis Meets Antibody Production: A Review. Antibodies 4:12-33.

Yin G., Garces E.D., Yang J., Zhang J., Tran C., Steiner A.R., Roos C., Bajad S., Hudak S., Penta K., Zawada J., Pollitt S., Murray C.J. 2012. Aglycosylated antibodies and antibody fragments produced in a scalable in vitro transcription-translation system. mAbs 4(2):1-9.

Zemella A., Thoring L., Hoffmeister C., Kubick S. 2015. Cell-free protein synthesis: pros and cons of prokaryotic and eukaryotic systems. ChemBioChem 16:2420-2431.

 

Topics: Cell-free technology

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