Youngthong, Chalermkiart; (2024) Establishing yeast Komagataella phaffii as an expression platform for prokaryotic nanocompartments and its potential application for antigen display. Doctoral thesis (Ph.D), UCL (University College London).

Text Final Post viva Chalermkiart Youngthong-18141345-PhD Thesis.pdf - Other Access restricted to UCL open access staff until 1 September 2025. Download (46MB)

Abstract

Prokaryotic nanocompartments, called encapsulins, are self-assembling protein structures (24-42 nm) found throughout bacterial and archaeal phyla. Prokaryotic encapsulins exhibit attractive attributes for bioengineering. Their shell exterior can be decorated allowing them to serve as delivery vehicles and vaccine candidates. Characterisation and engineering efforts have so far been restricted to bench scale and have predominantly been carried out in E. coli. However, to take candidates forward into industrial applications, effective production processes need to be established. In this work, we aim to establish methylotrophic yeast K. phaffii as an alternative expression platform with the goal of developing robust bioprocesses for encapsulin candidates of interest. Three encapsulins from Myxococcus xanthus, Synechococcus elongatus, and Thermotoga maritima (MxEnc, SeSrpI and TmEnc) were selected as candidates, and genetically designed to have the MycHis tag at their C-terminus for purification. These encapsulins were challenging to produce and purify in our standard E. coli expression system due to insolubility issues and low yield. Encapsulin expressions from K. phaffii resulted in soluble protein, reaching maximum yields at 48 h for MxEnc and SeSrpI shells and at 24 h for TmEnc shells. Intracellular encapsulins were subsequently recovered and purified from flask-scale productions. The MxEnc, SeSrpI and TmEnc yields were approximately 31.45, 0.52 and 0.1 mg/L, respectively. In term of particle attributes, self-assembling structures with two symmetries of T=3 (~32 nm) and T=1 (~20 nm) were observed from the MxEnc shell. The SeSrpI and TmEnc shells exhibited a uniform icosahedral structure corresponding to a T=1 symmetry with a size of ~23 nm, a small fraction of partially assembled shell observed from TmEnc. To functionalize our encapsulin candidate, we engineered a SeSrpI shell as a modular antigen display platform exploiting extended N- and C-arms on the surface. Two orthogonal Catcher proteins were individually and co-decorated on the shell exterior by fusing with two terminal ends. Three engineered SeSrpI candidates were intracellularly expressed into the soluble fraction with varying titers, and all candidates exhibited self-assembly. Subsequently, the SARS-CoV-2 receptor binding domain (RBD) with SpyTag secreted by K. phaffii was displayed on the SpyCat-SeSrpI shell where conjugated protein bands were observed. In summary, K. phaffii offers the capability to produce both native and engineered encapsulin shells, in which all candidates can be found in soluble fractions. Moreover, the surface displayed encapsulins produced by K. phaffii show promise as vaccine candidates. Therefore, K. phaffii has the potential to become an effective expression host if optimised further. Towards this goal, efforts will continue to determine how process parameters affect particle quality.

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