Recombinant Vector for Soluble FGF19 Expression
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Solution Overview
Problem
Current methods for producing recombinant fibroblast growth factor 19 (FGF19) face challenges such as insoluble expression and low yield due to its inherent structural characteristics, like amorphous structures and disulfide bonds, and the need for expensive proteases for separation from fusion partners, leading to immunogenic issues and reduced productivity.
Innovation Solution
A recombinant vector that includes a first polynucleotide encoding FGF19 and a second polynucleotide encoding disulfide bond isomerase (DsbC) is used, allowing for simultaneous and independent expression of synonymous codon substitution variants of FGF19 and DsbC, enhancing solubility and reducing the need for fusion partners, thereby improving expression levels and yield.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If FGF19 is expressed in E. coli, then production of recombinant protein is achieved, but the protein forms insoluble aggregates and cannot be expressed in soluble form
Solution Approach 1:
The patent introduces a fusion partner protein (such as GST, MBP, or Trx) that acts as an intermediary to enhance the solubility of FGF19. The fusion partner forms a soluble complex with FGF19, preventing aggregation while maintaining production levels. This mediator approach resolves the contradiction by allowing high expression without forming insoluble aggregates.
Solution Approach 2:
The patent creates a composite protein structure by fusing FGF19 with another protein domain (fusion partner). This composite construction combines the properties of both proteins - the production efficiency of FGF19 with the solubility enhancement provided by the fusion partner, thereby achieving both high expression and solubility simultaneously.
2Stability of the object's composition
If fusion partners are used to improve solubility, then soluble expression is achieved, but additional amino acids are added to FGF19 causing immunogenic problems
Solution Approach 1:
The patent applies protease recognition sequences (such as TEV protease, HRV 3C protease, or thrombin sites) to enable specific cleavage of the fusion partner from FGF19. This extraction approach allows the temporary use of fusion partners for solubility during expression, followed by precise removal to eliminate immunogenic sequences before final product formulation.
Solution Approach 2:
The patent performs preliminary purification steps including proteolytic cleavage and removal of fusion partners before the final product is used. This preliminary action eliminates immunogenic components in advance, ensuring the final FGF19 product is free from immunogenic sequences while having benefited from fusion partner-mediated solubility during production.
3Ease of manufacture
If proteases are used to separate FGF19 from fusion partners, then purification is achieved, but yield decreases and aggregation occurs during processing
Solution Approach 1:
The patent designs protease recognition sequences that enable highly specific and efficient cleavage at the fusion partner-FGF19 interface. This copying of natural protease sites ensures precise separation without requiring harsh conditions or excessive protease amounts, thereby minimizing aggregation and yield loss during the purification process.
Solution Approach 2:
The patent optimizes processing parameters including protease concentration, temperature, pH, and incubation time to achieve complete cleavage under mild conditions. By carefully controlling these parameters, the separation process maintains FGF19 solubility and prevents aggregation, preserving high yield while achieving effective purification.
4Stability of the object's composition
If wild type hFGF19 with disulfide bonds is expressed, then native structure is maintained, but expression in E. coli is difficult and results in inclusion bodies
Solution Approach 1:
The patent uses fusion partners with specific properties (such as GST or MBP) that act as mediators to facilitate proper folding and disulfide bond formation of FGF19 in the E. coli cytoplasm. These intermediaries provide a favorable microenvironment that supports native structure formation while preventing aggregation, enabling expression of structurally intact FGF19.
Solution Approach 2:
The patent replaces the need for complex oxidative folding environments with the use of fusion partners that inherently support proper disulfide bond formation in the reducing cytoplasm of E. coli. This substitution approach allows native structure formation without requiring periplasmic targeting or complex oxidation systems.
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
This approach results in increased soluble expression and higher yields of recombinant FGF19, overcoming the limitations of insoluble aggregation and immunogenic concerns, while maintaining the protein's functionality and stability.
Implementation Method 1
a second polynucleotide which encodes disulfide bond isomerase (DsbC)
Data Source
AI summary
A recombinant vector according to an embodiment of the present invention may produce fibroblast growth factor 19 (FGF19) having enhanced solubility. The synonymous codon substitution variant fibroblast growth factor 19 (scvhFGF19) and chaperone ΔssDsbC may be simultaneously and independently expressed in a host cell into which the recombinant vector is introduced, thereby it is possible to overexpress the fibroblast growth factor 19 in a soluble state.