Using Spontaneously Forming Ester Bonds to Engineer a Self-Assembling Enzyme Tree

Abstract

Spontaneously-forming intramolecular protein cross-links give rise to unique functions in nature and applications in biotechnology, and are often found in elongated bacterial cell surface adhesins. These isopeptide or ester bond cross-links in adhesin domains provide exceptional protein stability in a hostile extracellular environment. The bonds form between the first and last β-strand of the adhesin domains, and have been engineered for biotechnology applications requiring protein ligation. These ligation properties are engineered by removing the last strand from the adhesin; the two “split” parts when reintroduced, form a covalent cross-link. Whilst isopeptide technology is more developed, the ester bond equivalent has potential advantages. A greater sequence diversity in these proteins could provide numerous ligation domains all with specificity to their paired split domain partners. A multicomponent ligation system engineered with inherent specificity means that more complex structure assemblies could be achieved. This MSc project addresses the nature and potential of ester bond forming domains as a relevant technology. The primary objective was to engineer multiple split ester bond domains and to use them as a proof of concept application in biotechnology, specifically, to create a modular scaffolding system for the capture and clustering of enzymes, for increased metabolic efficiency. This project builds on a recently published ester bond ligation domain developed at the University Auckland, and on preliminary work showing ordered assembly of multiple ligation domains. The scaffold, referenced throughout this thesis using a tree analogy, uses a primary ‘trunk’ assembly with ‘branch’ domains to capture four green fluorescent proteins as cargo, analogous to ‘leaves’. In creating this system, 20 novel split ester bond domains were produced from numerous bacterial adhesins, and their ligation potentials characterised by SDS-PAGE and small-angle X-ray scattering. In parallel, another MSc student Robert Kerridge produced another set of 20 novel split ester bond domains. When combined, and as described in the final chapters of this thesis, a 13-protein scaffold was formed with captured GFP proteins. This breakthrough paves the way in the future for the development of scaffolded enzymatic pathways with high catalytic efficiency for use in applications such as biological batteries, enzymatic factories, and biosensors.