Applications of cell free protein synthesis in protein design

Abstract

In protein design, the ultimate test of success is that the designs function as desired. Here, we discuss the utility of cell free protein synthesis (CFPS) as a rapid, convenient and versatile method to screen for activity. We champion the use of CFPS in screening potential designs. Compared to in vivo protein screening, a wider range of different activities can be evaluated using CFPS, and the scale on which it can easily be used-screening tens to hundreds of designed proteins-is ideally suited to current needs. Protein design using physics-based strategies tended to have a relatively low success rate, compared with current machine-learning based methods. Screening steps (such as yeast display) were often used to identify proteins that displayed the desired activity from many designs that were highly ranked computationally. We also describe how CFPS is well-suited to identify the reasons designs fail, which may include problems with transcription, translation, and solubility, in addition to not achieving the desired structure and function.

Keywords: cell‐free protein synthesis; medium throughput; protein design; screening; surface immobilization.

FIGURE 1

Schematic representing the identification of functional protein designs. A large number of potential protein designs are created computationally. These designs are ranked and the top designs identified. These computationally top‐ranked designs are tested in CFPS—for production and activity. The proteins that display the highest activity in CFPS are then studied further, for example by purification and biophysical characterization.

FIGURE 2

Examples of protein functionality that can be assessed using CFPS. In each example, we show in cartoon form how three different designed proteins would behave in the assay. We represent more functional proteins by darker shades of blue. (a) Designing a transcriptional repressor to bind to a small molecule inducer. In this case, designed proteins are screened for their ability to bind the inducer (yellow). Proteins that function as desired are identified by an increase in GFP production in response to the presence of the small molecule. (b) Identifying designed proteins with increased enzymatic activity via production of a colored or fluorescent product. (c) Screening for binding. Protein variants that bind to another molecule (gray square) are identified via proximity induced fluorescence of the beads to which the protein and molecule are attached, in an AlphaLISA® assay. Other methods of detection, such as FRET, could also be used. (d) In some cases, detection of enzyme activity is only possible by removing and analyzing a sample from the CFPS, such as by gas chromatography (GC).

FIGURE 3

Examples of surface capture strategies from CFPS. (a) A His‐tagged protein produced by CFPS is captured to a Ni‐NTA coated surface. (b) Biotinylated DNA is bound to an avidin coated surface. CFPS from this DNA produces the protein product fused to GST (glutathione S‐transferase), which can then be captured to the surface by anti‐GST antibodies. (c) BslA‐SpyTag forms a self‐assembling protein monolayer. SpyCatcher‐protein fusion made by CFPS is captured via a covalent bond to the SpyTag of the BslA‐SpyTag surface coating.

FIGURE 4

Schematic illustration of the different points at which a protein design may fail. The point of failure can be identified using CFPS.