Figure 2.

Comparative overview of the characteristics associated with the major gene expression systems

Currently, the most commonly used protein production systems are E. coli, yeast, mammalian cells, and insect cells. In mammalian cells, both TGE and stable cell lines are frequently used, whereas in insect cells baculovirus-mediated expression is the predominant method of choice. The main characteristics associated with protein production in these systems are ranked on a scale of 1–5, which allows for an easy comparison of the individual characteristics between the different gene expression systems. The results presented here are based on a survey, which was organized among the members of the Protein Production and Purification Partnership in Europe (P4EU) network. The scores are weighted averages calculated from the survey responses. The survey first queried about the different gene expression systems used in the participants’ home laboratories. These data then formed the basis for deciding on the most commonly used gene expression systems in the community. Next, the participants were asked to score the individual characteristics associated with the gene expression systems they were familiar with based only on their own personal experiences (not on textbook knowledge). Sixty complete responses to the survey were received, which might seem like a small number of participants, but which in reality corresponds to a cumulative experience with thousands of different expression constructs. The characteristics that were assessed in the survey were: (i) ease of use, indicating how much experience/training is necessary to use a particular gene expression system; (ii) speed, which is the time required from plasmid DNA/expression construct to biomass (expressed protein) for processing; (iii) protein production capacity, which represents the average intracellular protein production capacity in mg/L of culture; (iv) protein secretion, which is the average range of secreted protein production capacity in mg/L of culture (secretion to the periplasm for E. coli, secretion to the extracellular milieu for yeast, mammalian, and insect cells); (v) protein folding and assembly related to the size of the protein(s) of interest, representing the ability to produce functional and correctly folded single-chain multi-domain proteins or multi-subunit protein complexes depending on their respective maximum size; (vi) protein folding and assembly related to the number of disulfide (SS) bonds, indicating the ability to produce functional and correctly folded (secreted) proteins depending on their respective number of disulfide bonds; and (vii) cost efficiency, estimating the consumable costs (e.g., media, transfection reagents, disposable flasks, plasmid preparation, cell maintenance, virus production, cell counting, etc.) for a 1-L production. All criteria are scored in a positive way, meaning higher scores correspond to more beneficial outputs.