Directed co-evolution of interacting protein–peptide pairs by compartmentalized two-hybrid replication (C2HR)

Abstract

Directed evolution methodologies benefit from read-outs quantitatively linking genotype to phenotype. We therefore devised a method that couples protein–peptide interactions to the dynamic read-out provided by an engineered DNA polymerase. Fusion of a processivity clamp protein to a thermostable nucleic acid polymerase enables polymerase activity and DNA amplification in otherwise prohibitive high-salt buffers. Here, we recapitulate this phenotype by indirectly coupling the Sso7d processivity clamp to Taq DNA polymerase via respective fusion to a high affinity and thermostable interacting protein–peptide pair. Escherichia coli cells co-expressing protein–peptide pairs can directly be used in polymerase chain reactions to determine relative interaction strengths by the measurement of amplicon yields. Conditional polymerase activity is further used to link genotype to phenotype of interacting protein–peptide pairs co-expressed in E. coli using the compartmentalized self-replication directed evolution platform. We validate this approach, termed compartmentalized two-hybrid replication, by selecting for high-affinity peptides that bind two model protein partners: SpyCatcher and the large fragment of NanoLuc luciferase. We further demonstrate directed co-evolution by randomizing both protein and peptide components of the SpyCatcher–SpyTag pair and co-selecting for functionally interacting variants.

Thermostability enhancement of polyethylene terephthalate degrading PETase using self‐ and nonself‐ligating protein scaffolding approaches

“…cPETase showed a biphasic melting profile with T m s of 44.5°C and 76.5°C (Figure 3). The first T m corresponds to PETase domain unfolding, while the second peak likely derives from unfolding of the SC domain thermostabilized by covalently bound ST (Siau et al, 2020). A similar observation has been reported for SC‐ST mediated cyclization of β‐lactamase enzyme (Schoene et al, 2014), indicating that the scaffold is likely promoting refolding of PETase upon heat‐denaturation instead of increasing resistance to unfolding.…”

Directed evolution of orthogonal RNA–RBP pairs through library-vs-library in vitro selection

“…To date, in vitro and E. coli or yeast cell-based in vivo library-vs-library selections have been reported to identify protein-protein ( 21–25 ), protein-peptide ( 26 ), peptide-peptide ( 27 , 28 ), and small molecule-protein ( 29 , 30 ), small molecule-RNA ( 31 ) pairs. This study is the first library-vs-library selection for RNA-protein interactions.…”

“…While these in vitro selection technologies usually involve the selection of an RNA or protein library for binding to a single target, an alternative approach is library-vs-library selection (or screening) which has been employed for identification of protein–protein ( 21–25 ), protein–peptide ( 26 ), peptide–peptide ( 27 , 28 ), small molecule–protein ( 29 , 30 ) and small molecule–RNA interactions ( 31 ). To our knowledge, however, RNA library-vs-protein library selection has not yet been reported.…”

Deploying synthetic coevolution and machine learning to engineer protein-protein interactions

“…In vitro, co-selection by mixing separate libraries is limited by the inability to isolate discrete coevolved pairs from complex mixtures, thereby losing connectivity between the sequences of members of interacting pairs ( 8 ). Coevolution studies that use in vivo functional selections such as bacterial in vivo screening ( 11 , 12 ) or yeast two-hybrid systems ( 13 ), as well as in vitro screening strategies including yeast mating systems ( 9 , 10 ) or compartmentalized two-hybrid systems ( 14 ), have been reported, but these systems are limited by small library sizes resulting in acquisition of sparse information rather than a broad evolutionary spectrum.…”