Incorporation of Lysine Tags as a Universal System for Controlling Protein Expression

Value Proposition

·        Precise and Tunable Protein Translation Control: Poly(A) track length can be varied from 1-12 AAA codons, providing precise and adjustable control over protein output.

·        Minimal Complexity: This technology offers predictable, graded control of protein expression using only a short DNA sequence insert—no external inducers, regulatory proteins, or complex genetic circuits required.

·        Versatility across Biological Systems: Works across bacterial and eukaryotic systems without species-specific optimization, reducing development time and costs when moving between expression hosts.

·        Dual Control: Unlike traditional expression control methods, this system uniquely modulates both protein output and mRNA stability, providing more comprehensive control over gene expression dynamics.

·        Compact and Modular: The small genetic footprint (up to 36 nucleotides) makes it easy to incorporate into existing constructs without disrupting other regulatory elements or significantly increasing plasmid size.

·        Overcomes Genetic Editing Limitations: Enables stable expression of toxic proteins at sub-lethal levels (something on/off systems can't achieve) by enabling precise intermediate expression levels

·        Standardized Research Tool: Codon-defined sequences provide a reproducible platform to probe ribosome stalling and mRNA stability mechanisms across different biological systems.

Unmet Need

Current methods for controlling protein expression lack precision, simplicity, and universality. Existing approaches suffer from environment dependent outcomes, require additional components or regulatory proteins, and often need extensive re-optimization when moving between different biological systems. These limitations are particularly significant in metabolic engineering, recombinant protein production, synthetic biology, and research applications. Therefore, there is a critical need for a simple, reliable, and universally applicable method to fine-tune protein expression at the translational level—one that works across all organisms without additional factors, enabling stable expression of essential and toxic proteins while preserving cell viability.

Technology Description

Researchers at Johns Hopkins have developed a simple and reliable method to control for protein production at the level of translation by incorporating poly(A)-encoded poly-lysine sequence tracks (consecutive AAA codons which encode for the amino acid lysine) within genes of interest. These sequence tags cause ribosome stalling during translation, creating tunable control where tag length (1-12 AAA codons) directly correlates with the degree of protein reduction—longer tags result in greater expression suppression.

Stage of Development

The system has been validated in both bacterial and eukaryotic cells

Data Availability:  n/a

Publications:

1. Laura L. Arthur et al. ,Translational control by lysine-encoding A-rich Laura L. Arthur et al. ,Translational control by lysine-encoding A-rich sequences.Sci. Adv.1,e1500154(2015). DOI:10.1126/sciadv.1500154
2. Kristin S Koutmou, Anthony P Schuller, Julie L Brunelle, Aditya Radhakrishnan, Sergej Djuranovic, Rachel Green (2015) Ribosomes slide on lysine-encoding homopolymeric A stretches eLife 4:e05534. https://doi.org/10.7554/eLife.05534 
3. Arthur LL, Chung JJ, Jankirama P, Keefer KM, Kolotilin I, Pavlovic-Djuranovic S, Chalker DL, Grbic V, Green R, Menassa R, True HL, Skeath JB, Djuranovic S. Rapid generation of hypomorphic mutations. Nat Commun. 2017 Jan 20;8:14112. doi: 10.1038/ncomms14112. Erratum in: Nat Commun. 2017 Feb 16;8:14705. doi: 10.1038/ncomms14705. PMID: 28106166; PMCID: PMC5263891.
4. Powell G, Pavlovic Djuranovic S, Djuranovic S. Gene dosage effects of poly(A) track-engineered hypomorphs. Mol Ther Nucleic Acids. 2021 Oct 8;26:865-878. doi: 10.1016/j.omtn.2021.10.005. PMID: 34729253; PMCID: PMC8536507.
5. Issued Patent: 11,603,533