More than just letters – the impact of protein sequence variation on expressibility and host cell responses

Background.

Yeasts such as Saccharomyces cerevisiae or Komagataella phaffii (syn. Pichia pastoris) are efficient hosts for recombinant protein production, both for biotherapeutics and industrial enzymes (Barone et al. 2023; Rettenbacher et al. 2022). Many of them are produced in a secretory manner. Production levels of such proteins range from µg to several g per liter, and it is very hard to predict a priori whether a protein will be well or hardly produced. Even for very closely related proteins (e.g. such obtained by protein engineering or isoforms from different species), production levels can differ significantly.
It is hypothesized that in eukaryotes, secretion yields are related to stability, with less stable proteins being more prone to misfolding and subsequent degradation rather than successful secretion. Indeed, it has already been experimentally verified in the late 1990s that for some proteins, such as bovine trypsin inhibitor (BPTI) expressed in S. cerevisiae, secretion yields were inversely correlated with thermodynamic stability (Kowalski et al. 1998). However, many of the previously investigated instable proteins were prone to aggregation, which is a known factor to trigger ER-quality control mechanisms such as ERAD (Sun & Brodsky, 2018).
While we agree that there is a correlation between stability and protein yields, we have evidence that thermodynamic stability is not the only determinant affecting the expressibility of proteins. For example, the introduction of stability enhancing mutations led to higher activity, but not higher amounts of secreted recombinant protein. This also becomes obvious when looking at the published variants, where usually only the most and least stable variants cause the strong correlation. Foldability and the abundance of certain amino acids are among the discussed factors (e.g. Listov et al. 2022; van den Berg. 2012).

Many of these studies were limited by the number of variants that were experimentally screened. Yeast surface display (YSD) is a valuable tool for high-throughput (HTP) screening of protein libraries established in both S. cerevisiae and K. phaffii (van Deventer et al. 2015; Kang et al. 2022; Stadlmayr et al. 2010), and the displayed protein levels usually correlate well with the amounts of secreted soluble protein (Shusta et al. 1999; Laurent et al. 2021).

Research questions.

• In which ways do variations in protein sequence (e.g. such as introduced through protein engineering) affect expressibility of such proteins in yeast?
• Which cellular pathways are affected if very closely related proteins (engineered protein variants vs. wild-type proteins) are expressed in yeast?
• Is there a correlation between expressibility and physico-chemical properties that goes beyond thermodynamic stability?

Project plan and methods.

Mutants of model enzymes and binders will be analysed regarding their secretion yields, stress responses and biophysical properties. Initially, available data and existing mutants will be investigated for their thermodynamic stability, stress response and secretion yields. To enlarge the design space, additional mutations will be introduced into the model proteins through random mutagenesis or targeted approaches, the latter aiming to generate mutants with similar thermodynamic stability but altered properties. To obtain a high-throughput correlation of cell stress to protein sequence variations, different sequence variants will be expressed as YSD-protein sequence libraries in yeast cells that contain fluorescent genetically encoded biosensors for folding- and secretion-related cellular responses (e.g. UPR biosensor) and analysed by flow cytometry and fluorescence activated cell sorting. Selected variants will be analyzed in order to differentiate between mutations affecting folding/secretion and activity/binding properties, as well as the more in-depth cellular response to their expression.

REFERENCES
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Kowalski JM, Parekh RN, Mao J, Wittrup KD. 1998. Protein folding stability can determine the efficiency of escape from endoplasmic reticulum quality control. J Biol Chem. 273(31):19453-8. doi: 10.1074/jbc.273.31.19453.
Kowalski JM, Parekh RN, Wittrup KD. 1998. Secretion efficiency in Saccharomyces cerevisiae of bovine pancreatic trypsin inhibitor mutants lacking disulfide bonds is correlated with thermodynamic stability. Biochemistry. 37(5):1264-73. doi: 10.1021/bi9722397.
Listov D, Lipsh-Sokolik R, Rosset S, Yang C, Correia BE, Fleishman SJ. 2022. Assessing and enhancing foldability in designed proteins. Protein Sci. 31(9):e4400. doi: 10.1002/pro.4400.
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van den Berg BA, Reinders MJ, Hulsman M, Wu L, Pel HJ, Roubos JA, de Ridder D. 2012. Exploring sequence characteristics related to high-level production of secreted proteins in Aspergillus niger. PLoS One. 7(10):e45869. doi: 10.1371/journal.pone.0045869.