An Improved Phage Display Strategy for Improving Protein Solubility
Dr. David Liu and his collaborators have developed a phage-based platform that can be used to rapidly improve the solubility of a given protein via continuous directed evolution. The platform features an ingenious genetic design that links successful
expression of the protein of interest (POI) to transcription of the pIII gene, which encodes the phage coat protein required for infectivity. To achieve this, Dr. Liu’s group utilized a T7 RNA polymerase split into two non-functional halves.
One half was tethered to the C-terminus of the ROI such that, in the event of failed or insoluble expression, the fully functional T7 RNA polymerase would not be assembled, leading to non-transcription of the pIII gene. This ensured that most
of the phage particles that failed to display sufficiently soluble POIs were automatically eliminated. When the selection system was applied to the direct evolution of a maltose-binding protein, however, it was discovered that frameshift mutations
could occur in a manner leading to accidental production of a functional, though mutated, half of the T7 RNA polymerase. The prevalence of such mutations increased with the selection stringency, and started to dominate the generated mutant
pool beyond a certain point. This problem can be rectified by introducing a secondary selection based on the enzyme activity of the ROI. Combined, the selection strategy demonstrated superior efficiency and led to the identification of a mutant
with five-fold better solubility and similar enzymatic activity within 3 days. The approach can also be applied to evolving other physicochemical properties such as thermostability.
