Recombinant protein expression is a time-consuming task because it requires the combination of a large number of variables that can often subvert timely development. Highly developed bacterial expression platforms are particularly suited to overcoming many of these obstacles because of the rapid growth rates of these organisms and the large number of expression tools that have been developed to meet expression challenges.
However, because primary amino acid sequences of large proteins reveal little information on how best to express a given protein, deciding which tools or combinations of tools will be effective to produce a particular target protein is often elusive. In particular, the expression of secreted proteins with multiple disulfide bonds can be challenging in a microbial system.
What has been needed is a way to apply parallel processing to expression strain development so that multiple expression strategies can be accessed and tested simultaneously. Pfenex has created a comprehensive Pseudomonas fluorescens-based protein production platform consisting of a broad spectrum of effective expression tools that can be seamlessly accessed and integrated to yield an expression strain producing large amounts of functional protein.
Replacing the traditional, linear, and iterative approach to strain development with a high-throughput, parallel-processing model enables construction and evaluation of 1,000 unique expression strains combining novel gene-expression strategies and host cell phenotypes in about one month’s time.
The toolbox pyramid diagram contained in Figure 1 describes the expression strategies and engineered hosts available through the platform that are used to generate thousands of completely unique expression strains. These tools can be seamlessly accessed so that any expression scenario can be combined with any engineered host by 96-well electroporation. Both the expression plasmids combining the expression strategies listed in Figure 1 and the competent host strains are off-the-shelf items.
The expression plasmids themselves all employ the same cloning strategy, utilizing optimized synthetic gene fragments to streamline target gene cloning. It is difficult to predict exactly which combination of secretion leader and ribosome binding site (RBS) will work best for the heavy chain (HC) and light chain (LC) of various Fabs.