Novel Proinsulin Glargine Structure for High-Yield Fermentation
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Solution Overview
Problem
Current methods for producing insulin glargine face challenges such as low yield and purity due to improper disulfide bond formation and miscleavage impurities, particularly in the use of copper/zinc superoxide dismutase fusion peptides and shortened C-peptide sequences, which complicate the recombinant protein preparation process.
Innovation Solution
A novel proinsulin glargine structure with an N-terminal fusion peptide sequence and a full-length human insulin Chain B, incorporating a '0 C peptide' strategy, is designed to improve folding and fermentation yield, using site-directed mutagenesis and specific amino acid sequences to enhance expression and purification in E. coli, followed by renaturation and trypsin digestion to obtain high-purity insulin glargine.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If copper/zinc superoxide dismutase fusion peptide is used for proinsulin expression, then expression level is improved, but miscleavage impurities increase and purity decreases
Solution Approach 1:
The patent removes the C-peptide sequence from the proinsulin structure, using only the A-chain and B-chain connected by disulfide bonds. This extraction of the problematic C-peptide region eliminates the source of miscleavage impurities while maintaining the functional insulin structure, thereby resolving the contradiction between expression level and purity.
Solution Approach 2:
The patent modifies the amino acid sequence by removing the C-peptide region, changing the structural parameters of the insulin molecule. This parameter change from full proinsulin to C-peptide-free insulin structure eliminates the miscleavage problem while preserving the essential disulfide bond connectivity and biological activity.
2Productivity
If shortened C-peptide sequence is used, then fermentation yield is improved, but miscleavage impurities increase
Solution Approach 1:
The patent completely removes the C-peptide sequence from the insulin structure, using only the A-chain and B-chain connected by disulfide bonds. This extraction eliminates the source of miscleavage impurities while maintaining the functional insulin structure, thereby resolving the contradiction between fermentation yield and purity.
3Reliability
If separate fermentation processes are used for chain A and chain B, then disulfide bond formation is improved, but process complexity increases and yield decreases
Solution Approach 1:
The patent combines the A-chain and B-chain into a single continuous polypeptide chain with the sequence B1-B30-A1-A21, eliminating the need for separate fermentation processes. The disulfide bonds are formed during a single expression and folding event, simplifying the overall process while ensuring proper disulfide bond formation through the natural folding pathway of the fused protein.
Solution Approach 2:
The patent segments the insulin molecule into A-chain and B-chain regions within a single continuous polypeptide, connected by a specific linkage sequence. This segmentation allows the chains to be expressed together as one protein while maintaining the ability to form correct disulfide bonds through controlled folding and proteolytic processing.
4Stability of the object's composition
If proinsulin with C-peptide is expressed, then folding is improved, but enzyme digestion efficiency decreases and yield is reduced
Solution Approach 1:
The patent removes the C-peptide sequence from the proinsulin structure, using only the A-chain and B-chain connected by disulfide bonds. This extraction simplifies the structure for more efficient enzyme digestion while maintaining proper folding through the essential disulfide bond connectivity, thereby increasing overall yield.
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
The approach increases the fermentation yield of insulin glargine by 75% or more, improves chromatographic purity to 99.4% or above, and reduces impurity content significantly, while also reducing production costs by optimizing enzyme digestion and purification processes.
Implementation Method 1
the sulfonated chain A and chain B in vitro to form an inter-chain disulfide bond
Implementation Method 2
the fusion peptide is separated from insulin glargine molecule by trypsinase digestion
Implementation Method 3
The precursor protein of the recombinant human insulin is synthesized by genetically modified organisms
Data Source
Figure 1~2
Figure 3
Figure 4A~4B
AI summary
The present invention discloses novel proinsulin glargine and a method for preparing insulin glargine therefrom, and belongs to the technical field of recombinant protein preparation. According to the present invention, a sequence of the proinsulin glargine containing SOD fusion peptide subjected to site-directed mutagenesis and "0 C peptide" is designed; recombinant Escherichia coli engineering bacteria for expressing insulin glargine are constructed; insulin glargine fusion protein in a form of an inclusion body is expressed by inducing the engineering bacteria; and denaturation, renaturation, modification, enzyme digestion, separation and purification are carried out to obtain a mature insulin glargine active pharmaceutical ingredient. According to the present invention, the SOD fusion peptide sequence is mutated to enhance the fermentation yield of the insulin glargine by 75%; and a "0 C peptide" strategy is adopted to avoid remaining of C-peptide residues and reduce the quality loss and miscleavage impurities in the enzyme digestion transformation. The purity of the insulin glargine active pharmaceutical ingredient prepared in the present invention is up to 99.9%, and the maximum single impurity content is controlled at 0.05%.