Insulin Aspart Fusion Protein Expression Yield
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Solution Overview
Problem
The existing methods for preparing insulin aspart are complex, technically difficult, and result in low yield due to the weak expression ability of Saccharomyces cerevisiae, increasing the cost of drug production and requiring a simpler, more environmentally friendly process with higher yield.
Innovation Solution
A fusion protein comprising a green fluorescent protein folding unit and an insulin aspart precursor is used, which is digested and purified through a series of chromatography steps to produce high-purity insulin aspart with correct protein folding and biological activity, including the use of Boc-modified insulin aspart and specific enzyme digestion conditions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If Saccharomyces cerevisiae is used as the expression host to produce insulin aspart precursors, then the preparation process can be established, but the expression ability is weak and the yield is low
Solution Approach 1:
The patent introduces a fusion protein as an intermediary carrier, where the insulin aspart precursor is fused with a carrier protein (such as glutathione S-transferase or alkaline phosphatase). This intermediary fusion protein can be expressed at high levels in E. coli, and the insulin aspart can be subsequently released through proteolytic cleavage, thereby solving the low yield problem caused by weak expression ability of Saccharomyces cerevisiae
Solution Approach 2:
The patent changes the expression system from Saccharomyces cerevisiae to E. coli, and modifies the protein structure by creating fusion proteins with specific carrier proteins. This parameter change in host organism and protein structure enables high-level expression and achieves a yield of 10-100 mg/L, significantly improving the productivity
2Manufacturing precision
If complex processes such as transpeptide are used to prepare insulin aspart, then the preparation can be completed, but the process becomes technically difficult and complex
Solution Approach 1:
The patent segments the preparation process into distinct functional modules: (1) expression of fusion protein in E. coli, (2) isolation of inclusion bodies, (3) proteolytic cleavage to release insulin aspart, (4) purification through chromatography. This segmentation simplifies each step and makes the overall process more manageable and scalable
Solution Approach 2:
The patent replaces complex chemical processes (such as transpeptide reactions) with biological processes (proteolytic cleavage by proteases). This substitution uses enzymatic mechanisms instead of complex chemical syntheses, thereby simplifying the preparation process while maintaining high purity
3Productivity
If maximum shaking culture of engineered bacteria is used, then the culture can be maintained, but the yield is limited to 21.5 mg/L which increases production cost
Solution Approach 1:
The patent performs preliminary action by fusing the insulin aspart precursor with a carrier protein before expression. This preliminary fusion enables the protein to be expressed at high levels in E. coli inclusion bodies, achieving yields of 10-100 mg/L. The subsequent release of insulin aspart through proteolytic cleavage is a straightforward process that maintains high yield while reducing production costs
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 method significantly increases the expression level and yield of insulin aspart, reduces production costs, and simplifies the purification process, achieving a purity of over 99% with improved bioactivity and environmental sustainability.
Implementation Method 1
FP is a green fluorescent protein folding unit
Implementation Method 2
digesting the insulation aspart fusion protein (primary protein) with enzyme, thereby obtaining a Mixture I containing Boc-modified insulation aspart
Implementation Method 3
purifying the Mixture II, thereby obtaining the insulation aspart
Data Source
AI summary
An insulin aspart derivative and a preparation method therefor are provided. The derivative has a fusion protein of a green fluorescent protein folding unit and an insulin aspart precursor or an active fragment thereof. The expression level of the fusion protein is significantly increased, and the insulin aspart precursor protein in the fusion protein is folded correctly and has a biological activity. In addition, the green fluorescent protein folding unit in the fusion protein can be digested into small fragments by a protease, which has a large molecular weight difference compared to the target protein and is thus easy to separate. Further provided is a method for preparing the insulin aspart and an intermediate by means of using the fusion protein.