DNA-Encoded Multivalent Display of Protein Tetramers on Phage: Synthesis and <i>In Vivo</i> Aplications
Bibliographic record
Abstract
Abstract Phage display links phenotype of displayed polypeptides with DNA sequence in phage genome and offers a universal method for discovery of proteins with novel properties. Injection of phage-displayed libraries in living organisms further provides a unique and powerful approach to optimize biochemical, pharmacological and biological properties of the displayed peptides, antibodies and other proteins in vivo . However, over 60% of the proteome is comprised of multi-domain proteins, and display of large multi-subunit proteins on phages remains a challenge. Majority of protein display systems are based on monovalent phagemid constructs but methods for robust display of multiple copies of large proteins are scarce. Here, we describe a DNA-encoded display of a ∼200 kDa tetrameric protein tetrameric L-asparaginase on M13 phage produced by ligation of SpyCatcher-Asparaginase fusion (ScA) to prospectively barcoded phage clones displaying SpyTag peptide. Starting from the SpyTag display on p3 minor coat protein or p8 major coat protein yielded constructs with five copies of ScA displayed on p3 (ScA 5 -phage) and 50 copies of ScA on p8 protein (ScA 50 -phage). ScA remained active after conjugation. It could be easily produced directly from lysates of bacteria that express ScA. Display constructs of different valency can be injected into mice and analyzed by deep-sequencing of the DNA barcodes associated phage clones. In these multiplexed studies, we observed a density-dependent clearance rate in vivo . A known clearance mechanism of L-asparaginase is endocytosis by phagocytic cells. Our observations, thus, link the increase in density of the displayed protein with the increased rate of the endocytosis by cells in vivo . In conclusion, we demonstrate that a multivalent display of L-asparaginase on phage could be used to study the circulation life of this protein in vivo and such approach opens the possibility to use DNA sequencing to investigate multiplexed libraries of other multi-subunit proteins in vivo . Abstract Graphic
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".