Biomanufacturability of Squid Ring Teeth Protein Library via Orthogonal High-Throughput Screening

Structural proteins are valued by materials engineers for their mechanical properties, chemical functionalities, and biodegradability. Several studies focus on high-throughput screening of structural proteins, which is crucial for understanding and improving proteins by analyzing entire sequence spaces, as demonstrated in directed evolution. A major challenge is the poor biomanufacturability of recombinant structural proteins, often due to toxicity issues (e.g., aggregation, cell stress, inclusion bodies) limiting yield. High-throughput screening can help solve these issues and improve the biomanufacturability of structural proteins. Based on naturally observed squid ring teeth proteins, we introduce a structural protein library, enabling us to explore a broad sequence space. We selected 33 amino acid fragments with four repeats from six different native squid species, creating a recombinant protein library of about 1.2 million variants (i.e., 33 ⁴ ). We demonstrated an orthogonal screening method that combines fluorescent- assisted cell sorting (FACS) and fluorescent microcapillary-array-based screening to establish correlations from genotype to single cells and ensembles. This optical high-throughput approach enables screening across the spectrum from individual cells to groups, facilitating the enrichment of desired clones. Our workflow distinguishes between expression and growth traits, supporting systematic genetic design studies focused on biomanufacturability. We observed that brighter clones tend to contain self-similar sequences (i.e., perfect repeats of fragments), whereas dimmer clones in the library have less similarity (i.e., imperfect repeats). The ability to screen large structural protein libraries not only accelerates research and development but also creates new opportunities for materials research and advanced biotechnological processes, underscoring its importance in modern synthetic biology.