A High-Throughput Approach
The Pfenex Expression Technology™ platform from Dow Chemical exploits the advantages of Pseudomonas fluorescens, including its ability to achieve high optical densities in both small- and large-scale fermentation, with high levels of specific protein expression and a high success rate relative to traditional E. coli and yeast-based fermentation systems. The scalable Pfenex platform reportedly enables the evaluation of more than 1,000 host strain/plasmid combinations within five weeks in parallel through the use of robotics to identify those strains producing the highest titer of soluble, properly folded, aglycosylated, active protein.
The platform is especially valuable for the rapid evaluation of “numerous putative antigens in late-stage discovery and/or preclinical studies in vaccine development,” says Patrick Lucy, global business development leader at Pfenex. He describes the toolbox of Pseudomonas-derived components Pfenex has developed for selecting production strain characteristics and fermentation conditions that optimize protein expression and folding.
These include a two-plasmid system—with one carrying the gene for the target protein and the other carrying genes coding for helper proteins, each under separate control (a complementation strategy that eliminates the need for antibiotics), a selection of ribosomal RNA binding sites to effect the rate of translation initiation, more than two dozen, well-characterized secretion leaders, and more than 150 unique protease deletion strains to address proteolysis.
In these engineered strains the protein of interest typically accumulates in the periplasm, where cleavage of the secretion leader and in vivo protein folding take place.
“We can rapidly screen over 1,000 strains in 96-well arrays using high-throughput screening methods,” says Lucy. The company recently implemented automated postexpression processing, including sonication to release the protein and subsequent sample handling and analysis of the antigens, which are ready for testing in animals. The platform has an “85% success rate for soluble protein expression for proteins that previously failed to express in other systems, and can produce milligram quantities of soluble antigen in eight to ten weeks,” Lucy adds.