Fusion Protein Structure for Peptide Solubility and Purification
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for synthesizing peptides, such as chemical synthesis and recombinant expression, face challenges with peptides less than 50 amino acid residues, including high costs, low yield, and inefficient purification due to large molecular weight and hydrophobicity, making it difficult to maintain affordable therapeutic peptide drugs and complicating the production of non-natural amino acid proteins like BOC-lysine.
Innovation Solution
A new fusion protein structure comprising a peptide bond or linker peptide with a β-folding unit of fluorescent protein, specifically designed to promote protein expression and yield, featuring a signal peptide and linker peptides that facilitate separation and purification, allowing for the expression and increased yield of target peptides and non-natural amino acid proteins.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If chemical synthesis is used to prepare peptides, then various therapeutic peptides can be prepared, but the process requires multi-step condensation, protection, deprotection, and purification steps, making it costly and generating large amounts of organic waste
Solution Approach 1:
The patent changes the synthesis approach from chemical condensation to recombinant expression, fundamentally altering the production parameters. This involves transitioning from amino acid fragment condensation to whole-gene expression in host cells, thereby eliminating the need for protection/deprotection steps and reducing organic waste generation
2Productivity
If fusion proteins with large molecular weight are used for peptide expression, then the target peptide can be expressed, but the fusion protein is difficult to separate due to strong hydrophobicity and large size
Solution Approach 1:
The fusion protein is designed with a cleavable linker region that segments the fusion protein into the target peptide and the carrier protein after expression. This segmentation allows for easy separation of the target peptide from the carrier through proteolytic cleavage, eliminating the purification difficulties associated with large hydrophobic fusion proteins
Solution Approach 2:
The patent introduces a linker peptide as an intermediary element between the target peptide and the carrier protein. This linker serves as a mediator that facilitates both expression and subsequent separation, allowing the target peptide to be expressed as part of the fusion protein while providing a cleavage site for easy release and purification
3Productivity
If traditional fusion proteins are used, then the target protein can be expressed, but the target protein has low specific gravity, low fusion ratio, stable structure, and is difficult to digest
Solution Approach 1:
The patent applies local quality by designing specific regions of the fusion protein with different properties: the carrier protein region provides stability and solubility, the linker region provides cleavability, and the target peptide region maintains its native structure. This localized functional differentiation resolves the contradiction between stable structure and ease of digestion
Data Source
AI summary
Provided are a fusion protein containing fluorescent protein fragments and uses thereof. The fusion protein according to the present invention has a structure as expressed by (P1-L1)s-A1-(X)n-A2-(L2-P2)t, wherein (X)n or A1-(X)n can promote the folding and expression of fused target peptide, improve the solubility of the fusion peptide and reduce the interaction among fusion peptide molecules. (X)n or A1-(X)n in the fusion protein can be cut by enzymes into a plurality of short peptides with a length much shorter than that of the target peptide, which facilitates the separation from the target peptide so as to make the purification of the target peptide more convenient.