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July 01, 2011 (Vol. 31, No. 13)

Achieving Optimal Expression for Fabs

Using a Combinatorial Approach in a P. fluorescens System to Boost Production

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    Figure 1. Strain screening process: 1,000 or more unique expression strains are constructed and screened to identify combinations that result in high levels of active Fab expression.

    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.

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    Figure 2. Rapid cloning vectors: monocistronic or bicistronic expression vector libraries employ a single cloning strategy for streamlined fusion of target-encoding genes with a variety of ribosome binding site strengths and periplasmic secretion leader coding sequences.

    In fact, we have observed that with each Fab expressed, the optimal strain selected for each Fab is composed of different expression strategy/host strain combinations. A number of monocistronic and bicistronic constructs is screened for any given Fab, which is facilitated by the rapid cloning vectors illustrated in Figure 2.

    Synthetic fragments designed with HC and LC coding regions, as illustrated in the figure, are cloned into any number of rapid cloning vectors, which are then electroporated into an array of host strains. These hosts may have one or more protease genes deleted and/or may overexpress folding modulators (chaperones, disulfide isomerases), which enable production of high levels of intact, assembled, functional Fab.

    The ability to perform all aspects of strain engineering in an automated, high-throughput, 96-well format is key to this combinatorial approach. An obligate aerobe, P. fluorescens, is uniquely suited to growth by simply shaking in a 96-well plate (0.5 mL culture volume) without oxygen supplementation, reaching cell densities of 30–50 A600 units, as described in Figure 1.

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