Understanding protein-body formation and purification

Background.

Protein bodies (PBs) are naturally occurring protein storage organelles/structures in a subset of differentiated plant cells, e.g. maize endosperm cells (Larkins and Hurkman 1978). They consist of several types of proteins, e.g. zeins, that form distinct layers within a PB and trigger specific events during PB formation, e.g. nucleation, growth, shell formation (Shewry et al. 1995; Woo et al. 2001; Lending and Larkins 1989; Mainieri et al. 2014). Even if the proteins constituting PBs are modified, e.g. through fusion to reporters like fluorescent proteins or functional moieties like antigen, the PBs can still form (Schwestka et al. 2023; Schwestka et al. 2020; Whitehead et al. 2014; Llop-Tous et al. 2010; Takaiwa et al. 2019; Takagi et al. 2010). Whereas, several stages of PB formation have been reported (Guo et al. 2013), the underlying mechanisms, i.e. time course, potential sources of PB size heterogeneity etc., are largely unknown at the moment. Nevertheless, PB formation can be induced artificially, for example through transient (co-)expression of the respective proteins in plants and even plant cell culture (Schwestka et al. 2023). In this respect, we have developed an automatable high-throughput method called ‘plant cell packs’ (PCPs) that facilitate the rapid screening of various protein variants and allows co-expression of proteins too (Rademacher et al. 2019; Gengenbach et al. 2020). Therefore, the PCP technology can be used to assess and understand the impact of, for example, plant (cell) cultivation conditions on PB formation. A time-resolved monitoring of PB formation in planta would greatly expand this understanding, but is difficult in intact plants and PCPs alike. Instead, microfluidic devices facilitate continuous single cell monitoring (Westerwalbesloh et al. 2019; Blöbaum et al. 2023) and would therefore be a useful asset in the analytical repertoire.
Due to their stability towards proteolytic degradation, the option to modify the zeins of which they are composed, and their size in the range of 0.5–2.0 μm, PBs can potentially be used as delivery vehicles for protein-based oral vaccines. However, PB size is also a major obstacle during purification because cell debris released during PB extraction from plants and plant cells is typically in this range too (Buyel et al. 2015), so that conventional filtration approaches result in enriched, but not pure PB fractions (Schwestka et al. 2020). As a result, PB purification currently relies on density gradient centrifugation-based separation (van Zyl et al. 2017; Hofbauer et al. 2016), which is time and resource intensive and hardly scalable. This is a substantial hurdle in the context of any attempt to scale-up PB production that is necessary for an indepth characterization and potential pharmaceutical applications. Importantly, efforts in the context of bionanoparticle (e.g. virus-like particle, exosomes etc.) production have resulted in novel purification tools including core-shell, fiber-based and monolithic chromatography resins (Kawka et al. 2021). In addition, novel types of membranes have emerged that could be combined with such resins to design a costefficient and scalable PB manufacturing.
Obviously, a prerequisite of developing such a manufacturing process is the detailed knowledge about PB properties, e.g. size distribution, zeta-potential and hydrophobicity (Carvalho et al. 2022).

Aims and methods.

In this thesis, the candidate will first characterize PBs in terms of their genesis in planta and build an understanding of physico-chemical PB properties and factors causing PB heterogeneity. Afterwards, the collected data will be used to design, test and optimize purification strategies for PBs in a rational, targeted manner.
Specifically, the student will use a PCP-based high-throughput screening system to transiently express zeins, triggering PB formation and then perform a coarse characterization of PB variability, e.g. in terms of plant cell age, incubation time, protein type and fusion partner and extraction conditions. Characterization will include microscopic analysis, dynamic light scattering and protein-chemical analysis after density gradient centrifugation-based purification. Extreme conditions of PB formation will be identified to capture heterogeneity using, for example, a principal component analysis. The extreme cases will be subjected to a detailed analysis using microfluidic devices that facilitate time-resolved microscopic observation of single cells in the course of PB formation.
Based on the characterization of PBs and their formation, the candidate will devise a set of scalable downstream processing options that can consist, for example, of i) PB concentration using membrane technologies (removing most plant host cell proteins in the process), ii) PB capture using fiber-based chromatography, iii) polishing using core-shell resins. Other options to be tested can include aqueous twophase separation, depth filters with large pores (>20 μm) or fractionated precipitation (e.g. using flocculants).
Lastly, the impact of purification processes on PB properties (stability, particle size distribution etc.) will be assessed.

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