Self-similar Sequences Yield Higher Protein Expression in a Squid Ring Teeth Protein Library

Protein materials give biological systems remarkable abilities, including tunable control over structural, optical, electrical, self-healing, and thermal properties. High-throughput screening of structural proteins is essential for understanding and enhancing proteins by exploring full sequence spaces, as shown in directed evolution. A significant challenge is the poor protein yield of recombinant structural proteins, often due to toxicity issues (e.g., aggregation, cell stress, formation of inclusion bodies), which limits protein yield. There is a need to address these issues and enhance the expression yield of structural proteins. Based on naturally observed squid ring teeth proteins, we introduce a structural protein library that allows us to explore a broad sequence space using a new high-throughput platform. We selected 33 amino acid fragments from six different native squid species and constructed a protein library containing every possible fusion of four such fragments (approximately 1.2 million variants, 33 ⁴ ). We analyzed subsets of this library using a multi-step screening method that combines fluorescent-assisted cell sorting (FACS) and fluorescent microcapillary-array-based screening to establish correlations between structural protein sequences in single cells and in clonal populations. Our workflow considers both protein expression and cell growth, supporting systematic genetic-design studies focused on protein expression yield. We observed that protein sequences with higher self-similarity tend to have greater expression levels. This suggests that self-similarity is a crucial design parameter for the heterologous expression of material-forming proteins with repetitive sequences, a factor that was not previously addressed. The ability to screen large libraries of structural proteins for expression and cell growth enables high-yield protein production, crucial for synthetic biology and biomanufacturing.