P69: Rapid Therapeutic Protein Production in Pseudomonas fluorescens Utilizing a High-throughput, Parallel Processing Approach

Pseudomonas fluorescens, a Gram-negative bacterium, has been specifically developed as a protein production platform to enable rapid identification of strains capable of expressing high titers of soluble and active therapeutic protein. Pfēnex Expression Technology^TM employs a toolbox of defined P. fluorescens host strains, including protease deficient mutants and chaperone/ foldase overexpressors combined with a set of expression vectors encoding a variety of transcription and translation regulators, as well as a collection of periplasmic secretion signals. High-throughput automated transformation, growth, and expression analysis at the 96-well scale was used to rapidly identify expression strategy and host strain combinations for production of high soluble yields of recombinant therapeutic proteins. Employing a combinatorial approach to strain engineering, thousands of unique expression strains are constructed and evaluated in approximately 5 weeks. Expression strain and host strain combinations identified at the 96-well scale were then selected for fermentation “range-finding” experiments to identify the most robust strains for production of larger quantities.  We have applied these methods to successfully produce difficult to express therapeutics including human G-CSF, antibody derivatives and vaccine antigens.