Codon-Optimized MG53 Polypeptides for Recombinant Expression
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Solution Overview
Problem
Current methods are inadequate for effectively modulating cell membrane repair processes, which are crucial for treating various diseases and conditions related to cellular and tissue damage, including neurodegenerative diseases, heart issues, and muscle disorders, as they fail to optimize the production and expression of therapeutic proteins involved in membrane repair.
Innovation Solution
The development of nucleic acids and polypeptides, specifically MG53 and its interacting proteins, which are optimized for expression and solubility using codon-optimized sequences, tags like histidine and thioredoxin, and modifications for enhanced bioavailability and stability, facilitating their use in therapeutic compositions for tissue repair and regeneration.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If conventional methods are used to produce therapeutic proteins, then production is possible, but expression levels and solubility are insufficient
Solution Approach 1:
The patent applies parameter changes by optimizing the nucleotide sequence through codon optimization tailored to specific host organisms (E. coli, CHO cells, HEK293 cells). This involves adjusting codon usage patterns, GC content, and sequence features to maximize protein expression levels and solubility in the target host system, directly resolving the contradiction between achieving high protein quantities and maintaining ease of manufacture.
Solution Approach 2:
The patent introduces intermediary elements including signal peptides, fusion tags (His-tag, MBP, GST, Trx), and protease cleavage sites that facilitate protein production, solubility enhancement, and purification. These intermediaries mediate between the genetic code and the functional protein, enabling efficient manufacture while maintaining high expression levels and solubility.
2Stability of the object's composition
If therapeutic proteins are produced without optimization, then production is achievable, but solubility and stability are inadequate
Solution Approach 1:
The patent employs solubility-enhancing fusion tags such as maltose binding protein (MBP), glutathione S-transferase (GST), and thioredoxin (Trx) as intermediary elements. These tags act as mediators that prevent protein aggregation and enhance solubility while maintaining production efficiency. The tags are subsequently removed via protease cleavage, leaving the stable, soluble therapeutic protein.
Solution Approach 2:
The patent modifies protein stability parameters by optimizing the coding sequence to enhance folding, reducing aggregation-prone regions, and incorporating disulfide bond-forming cysteines where appropriate. These parameter changes in the nucleotide and protein sequence directly improve solubility and stability without compromising production efficiency.
3Ease of manufacture
If recombinant proteins are produced in host cells, then protein synthesis is achieved, but recovery and purification are difficult
Solution Approach 1:
The patent introduces affinity tags such as polyhistidine (His-tag) sequences as intermediary elements that facilitate protein recovery and purification. These tags serve as mediators between the protein and purification media, enabling simple affinity chromatography-based recovery processes. The tags are positioned at the N- or C-terminus and can be removed enzymatically after purification, simplifying the overall process.
Solution Approach 2:
The patent segments the protein production process into distinct functional modules: expression module (with optimization features), purification module (with affinity tags), and final protein module (without tags). This segmentation allows each component to be optimized independently, making recovery and purification easier while reducing overall process complexity.
Data Source
AI summary
Disclosed herein are nucleic acid sequences that encode novel polypeptides. In particular, the present invention provides nucleic acid molecules that include optimization features that enhance the expression and/or recovery and/or activity of encoded polypeptides. Also disclosed are polypeptides encoded by these nucleic acid sequences, and antibodies, which immunospecifically-bind to the polypeptide, as well as derivatives, variants, mutants, or fragments of the aforementioned polypeptide, polynucleotide, or antibody. The invention further discloses therapeutic, diagnostic and research methods for diagnosis, treatment, and prevention of disorders involving any one of these novel human nucleic acids and proteins.