Fab

Periplasmic expression, multiple secretion leaders:
8 expression plasmids x 31 expression hosts=
248 strains tested in parallel
96 well format, 0.5 ml cultures

Protein leads such as Fab fragments are first expressed in hundreds of unique host strains combining elements of the Pfēnex toolbox. Predicting which components of the toolbox will be crucial in successful expression of any given protein cannot be determined from intrinsic information, such as amino acid sequence. This means that the combination of critical factors is empirical for each individual target protein. Accordingly, Pfēnex Inc has developed a high throughput screening work process that enables the parallel evaluation of hundreds of unique host strains containing a variety of expression strategies for a specific gene. When designing a strain engineering and screening strategy, Pfēnex scientists utilize an algorithm to evaluate certain attributes of the protein to be expressed such as the presence of cysteine residues, molecular weight, propensity for proteolytic degradation, and expression performance in other hosts. In the case study illustrated here it is evident that both host strain phenotype and the choice of secretion leader for the heavy and light components of the Fab fragment impact the amount and activity of the intact, active Fab. Further, fermentation scouting and range-finding in scaled down equipment, varying a number of fermentation parameters (using a Design of Experiments, DOE approach) is shown to increase the level of expressed active Fab fragment by between 10 and 20-fold. These results emphasize that both components, strain engineering and fermentation DOE are necessary to reach the desired outcome.

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