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

Jia Wei Siau, Samuel Nonis, Sharon Chee, Li Quan Koh, Fernando Ferrer, Christopher Brown, Farid Ghadessy

Research output: Contribution to journalArticlepeer-review

4 Citations (Scopus)

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.
Original languageEnglish
Article numbere128
Pages (from-to)E128
Number of pages14
JournalNucleic Acids Research
Volume48
Issue number22
DOIs
Publication statusPublished - 16 Dec 2020
Externally publishedYes

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