Small-scale Strain Evaluation
It is important that cell growth and expression during the strain-development stage be scalable. Scaled-down growth of P. fluorescens in 96-well plates was developed as a simple process, using a defined minimal salts medium without animal-derived components. Antibiotics are not required for plasmid maintenance during strain development steps or scale-up, as plasmids are maintained by complementation.
Growth of P. fluorescens in 96-well plates (0.5-mL culture volume in a 2-mL deep well block) performed in standard shakers with no oxygen enrichment was found to reach cell densities similar to that obtained in shake flask culture, usually 30–50 optical density units (Figure 1).
The ability to achieve such high cell densities allows for production and recovery of active protein rapidly at small scale, which can be advantageous for screening activity of multiple proteins as well as for expression of a particular recombinant protein in an array of P. fluorescens host strains. High-throughput transformation and expression of several recombinant therapeutic proteins can be readily performed using an array of host strains.
As an example, human gamma-interferon (g-interferon), which is typically expressed as insoluble protein in E. coli unless fused to another protein such as NusA, was expressed in 90 P. fluorescens host strains. Previously it was shown that soluble, active g-interferon can be expressed in wild type P. fluorescens. Within two weeks, strains were transformed with a g-interferon-encoding plasmid and analyzed for the expression of soluble target protein.
The soluble portion of cell extracts was analyzed in high-throughput format using 96-well SDS-CGE. This tool is useful as a first-line method to estimate soluble target protein expression and to rank the productivity of a number of strains. Several host strains were identified that resulted in improved soluble g-interferon expression when compared to soluble g-interferon expression in the wild type strain (Figure 2).
A variety of host strains identified showed significant improvement of g-interferon expression. We observed that growth and expression results obtained through high-throughput expression analyses are typically reproducible at the 20-L fermentation scale. That is, strains exhibiting good growth and expression of recombinant target protein at the high-throughput scale generally produce high levels of quality, active protein when grown at the 20-L fermentation scale. The ability to screen multiple host strains in high-throughput format and scalability of those results greatly reduces expression strain development time, allowing faster progression of a therapeutic protein to the clinic.