When protein engineers think of surface display, they almost invariably think of antibody engineering. But to limit these powerful platforms to antibodies is to overlook the fundamental theory that any protein of interest can be displayed on cell surfaces. This includes enzymes and receptors.
Engineering Enzymes: Screening for Catalysis, Not Just Binding
The fundamental challenge with enzyme engineering via surface display is measuring a dynamic catalytic reaction within the static snapshot of a FACS experiment. The solution is to convert enzymatic activity into a stable fluorescent signal on the cell surface.
Strategies for Capturing the Signal at the Cell Surface
1. Leveraging Intrinsic Product Properties
If the enzyme cleaves a substrate to reveal a charged or hydrophobic moiety, the product associates with the cell surface. This approach works well with fluorogenic substrates.
2. Biotin-Streptavidin Capture
The substrate is synthesized with a biotin tag. The product stays localized on the surface, and fluorescently-labeled streptavidin “paints” the active cells for sorting.
3. Substrate Tethering
The substrate is chemically cross-linked to the cell surface. The enzyme acts on the substrate attached to its own cell, enabling covalent retention of the fluorescent product.
4. Droplet Encapsulation
Cells are encapsulated in picoliter water-in-oil droplets using microfluidics. Each droplet functions as a microscopic test tube. The fluorescent product accumulates within the droplet, and droplets are sorted by FACS.
Engineering Receptors: Screening for Function Beyond Affinity
Affinity and Specificity
Receptor affinity screening is analogous to antibody screening. Counter-screening with dual fluorophores (red target + green off-target) allows collection of red-positive/green-negative cells for specificity selection.
Enhanced Stability and Expression
Sorting for the brightest cells using an anti-expression-tag antibody enriches for improved folding and expression. This can be combined with heat or pH stability challenges to select for robustness.
Downstream Signaling
For receptors like GPCRs, the signaling cascade can be converted into a fluorescent signal using reporter genes (e.g., GFP downstream of the receptor’s signaling pathway).
Related Ranomics services
- Enzyme engineering: Directed evolution and display-based selection for enzyme activity and stability.
- Receptor targeting: Engineering receptors and receptor-directed binders on display platforms.