Polynucleotide Construct Tuning Secreted Surface-Bound Polypeptide Ratio
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Solution Overview
Problem
Current methods for co-expression of membrane-bound and secreted polypeptides in desired ratios face inefficiencies, particularly when using 2A elements in non-native sequences, which can reduce cleavage efficiency and affect the production of specific ratios of secreted and surface-bound proteins.
Innovation Solution
A polynucleotide construct comprising a signal sequence, a cleavage site encoded by a 2A polypeptide, and a membrane anchor, arranged in a specific order to facilitate controlled separation and localization of polypeptides, allowing for tunable expression of secreted and surface-bound proteins through a 'molecular rheostat' system.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If 2A elements are introduced into non-native sequences, then the polynucleotide construct can be flexibly designed, but cleavage efficiency decreases
Solution Approach 1:
The patent applies parameter changes by systematically mutating specific amino acid residues at the 2A element cleavage site to optimize cleavage efficiency. By altering parameters such as residue identity, position, and surrounding sequence context, the invention achieves high cleavage efficiency (>90%) in non-native sequences while maintaining design flexibility. This resolves the contradiction by finding optimal parameter combinations that satisfy both adaptability and reliability requirements.
2Productivity
If cleavage efficiency is increased, then the ratio of secreted to surface-bound polypeptides is improved, but the ability to tune expression ratios is reduced
Solution Approach 1:
The patent implements dynamics by creating a tunable cleavage site system where the cleavage efficiency can be dynamically adjusted by selecting different 2A element variants or modifying specific residues. This allows the ratio of secreted to surface-bound polypeptides to be controlled across a range (e.g., 1:1 to 10:1) while maintaining high overall productivity. The system transforms a static cleavage mechanism into a dynamic,可调 parameter that adapts to different expression requirements.
3Productivity
If multiple polypeptides are co-expressed from a single mRNA, then protein production efficiency is improved, but the complexity of controlling individual polypeptide ratios increases
Solution Approach 1:
The patent applies segmentation by dividing the single polypeptide product into multiple separate polypeptides through the 2A element-mediated cleavage mechanism. By incorporating multiple cleavage sites with different efficiencies within a single mRNA construct, the system can generate multiple polypeptides (e.g., membrane-bound and secreted forms) with controlled ratios. This segmentation approach maintains high productivity while simplifying control compared to using separate mRNA constructs for each polypeptide.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Enables precise control over the ratio of secreted to surface-bound polypeptides by selecting cleavage sites with specific activities, optimizing protein production and localization in target cells.
Implementation Method 1
the presence of the 2A element in the mRNA can cause the translating ribosome to undergo an intra-ribosomal, translational termination-and-restart event during the synthesis of the nascent polypeptide chains
Implementation Method 2
The presence of a signal sequence on the polypeptide can mediate the translocation of the polypeptide to the cell's endoplasmic reticulum
Implementation Method 3
If an anchor sequence is present on the protein or polypeptide, translocation into the endoplasmic reticulum can stop upon the entry of the anchor sequence into the pore, before the entire polypeptide is transported into the endoplasmic reticulum
Implementation Method 4
Cleavage of a luminal portion of a protein or polypeptide from a membrane-bound portion of the polypeptide in the endoplasmic reticulum, golgi, in a membrane vesicle, and/or at the cell surface
Data Source
AI summary
Some embodiments herein provide compositions and methods for expressing secreted and cell-surface-bound polypeptides in a single cell. In some embodiments, secreted and cell-surface polypeptide are produced from a single polynucleotide. The polynucleotide can comprise a sequence (or sequence encoding a polypeptide) that mediates separation of a membrane anchor from the polypeptide. In some embodiments, a desired ratio of secreted to surface-bound polypeptide is obtained by selecting a sequence that mediates a desired level of separation of the membrane anchor from the polypeptide.