ASU iGEM: OptoPACE

Protein engineering is the process of constructing proteins (enzymes) with altered properties or structures. Due to the importance and ubiquity of Proteins in every facet of life, scientists have increasingly turned towards proteins to describe functional bases of organisms and cells. Protein engineering has commonly focused on the idea of improving enzyme specificity or pushing catalytic activity from one substrate to a slightly altered one. However, this reflects an old idea, and with the advent of newer, more advanced modelling and computational techniques, Protein Engineering has expanded beyond the concept of functional evolution.

Although protein engineering is often described as being important for future therapeutics, the role of protein engineering is frequently described in a narrow scope: focused primarily on alternative scaffolds which act as antibody substitutes, fusion proteins for increased half-life, PEGylation, glycosylation, or mutagenesis for reduced immunogenicity. In reality, the impact of protein engineering will be much broader. The tuning and design of protein stimulus-responsive behavior is now possible through protein engineering and this has the potential to transform the drug metabolism landscape. In particular, the use of engineered proteins for targeted drug activation or neutralization offers the potential for significantly enhanced control of pharmacokinetics and pharmacodynamics, and for reduced side effects when used in combination with powerful anticancer therapeutics. (1)

References

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1. Protein Engineering: A New Frontier for Biological Therapeutics

Rational design methods focus on utilizing rigorous biochemical and physical modelling techniques to simulate possible changes and interactions between different elements in a closed system. Because this can be difficult with exponentially complex systems, these methods have always fallen aside compared to in vitro approaches. However, with the boom of ML and structural prediction models like AlphaFold, rational design has been catapulted back into the center square. Nowadays, these approaches focus on utilizing neural network architectures to learn physical properties and predict interactions in mutations.

Directed evolution methods focus on using natural fitness as a pressure for species to mutate. What this means is growing cultures of different organisms in increasingly uncomfortable conditions to enforce a selective pressure on the populations. In Protein Engineering, the common methods utilize phages to perform PACE, or phage assisted continuous evolution. This method uses selective pressures on the phages themselves rather than the target organism, and allows for a specificity to evolve a singular protein with one function. This method is largely demonstrated on transcriptional elements, in which the survival of Phage populations depends on the trajectory of evolution trending towards a desired result. A good example is the evolution of a T7 polymerase to have specificity and activity on T3 promoters. The paper is linked below:

Negative selection and stringency modulation in phage-assisted continuous evolution

Site Saturation Mutagenesis methods use error-prone cloning of proteins on locations that have been experimentally determined to either be structurally or functionally significant. These methods commonly focus on attempting to change the substrate affinity and activity of catalysts to induce a novel activity upon the protein. Upon finishing a cycle of Site Saturation Mutagenesis, researchers will often use the results to then seed the start of a Directed Evolution method, as a way to introduce diversity into the starting population without changing the fitness of the oerall population.