rhNGF Gene Combination Enhances Eukaryotic Expression Yield
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Solution Overview
Problem
Current eukaryotic expression systems for recombinant human nerve growth factor (rhNGF) face challenges in achieving high-efficiency expression due to unsatisfactory protein folding and low production yield, primarily because of the lack of effective post-translation modification functions, leading to intricate production processes and high costs.
Innovation Solution
A gene combination comprising specific regulation components, such as 'gene B' and 'gene C', is selectively connected to the human nerve growth factor pro gene to enhance expression in a eukaryotic expression system, specifically using CHO cells, which results in higher secretion efficacy and production levels of rhNGF.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If E. coli expression system is used to produce rhNGF, then production cost is reduced and production yield is increased, but the expression products form inclusion bodies requiring intricate in vitro renaturation process
Solution Approach 1:
The patent segments the production process by using E. coli to produce only the precursor form (proNGF) which naturally forms inclusion bodies, then separates the renaturation step as a distinct controlled process. This allows the high-yield bacterial expression to be decoupled from the complex folding requirements, managing complexity through process segmentation rather than elimination.
Solution Approach 2:
The patent applies preliminary action by producing proNGF with the signal peptide and pro-region already attached in the E. coli system. The inclusion body formation is anticipated and prepared for in advance, allowing subsequent standardized renaturation protocols to be applied. This preliminary structuring simplifies the overall process despite the apparent complexity of inclusion body handling.
2Productivity
If proNGF is expressed in E. coli system, then production yield is increased, but correct folding of mature NGF protein is compromised due to lack of post-translation modification function
Solution Approach 1:
The patent uses proNGF as an intermediary form that bridges the gap between bacterial expression capabilities and eukaryotic protein folding requirements. The pro-region and signal peptide act as mediators that guide proper folding during the controlled renaturation process, enabling high-yield bacterial production to result in correctly folded functional protein after the intermediary processing step.
3Reliability
If mouse nerve growth factor (mNGF) is used for injection, then treatment effectiveness is achieved, but safety issues and viral infection risk arise due to amino acid sequence differences and heterologous protein nature
Solution Approach 1:
The patent applies parameter changes by modifying the amino acid sequence parameters of NGF from mouse origin to human origin. The recombinant human NGF (hNGF) shares the same structural and functional parameters (118 amino acids, disulfide bond pattern, dimer structure) that provide treatment effectiveness, while changing the sequence identity parameter to eliminate immunogenicity and safety concerns associated with heterologous proteins.
Data Source
AI summary
A gene combination capable of high-efficiency expression of rhNGF is provided to optimize gene expression regulation components of a recombinant human nerve growth factor. The gene combination enhances expression of rhNGF. As shown by experiments, the gene combination is capable of high-efficiency expression of a recombinant human nerve growth factor (rhNGF) with natural activity in a eukaryotic expression system.