Recombinant MYDGF Expression With N-Terminal Degradation Protection
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Solution Overview
Problem
Current recombinant human myeloid-derived growth factor (MYDGF) production methods face challenges such as high costs, degradation issues, and potential antigenicity risks, particularly in mammalian expression systems, leading to impurities and unsuitability for clinical use.
Innovation Solution
Development of recombinant MYDGF proteins with minimal degradation and post-translational modifications, produced in prokaryotic systems, maintaining the native structure and reducing antigenic epitopes, allowing scalable production up to 300 g per batch.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If recombinant human MYDGF is produced in mammalian expression systems (HEK-293T or CHO cells), then the protein can be produced with proper folding and structure, but the production costs become excessively high and the protein undergoes significant degradation
Solution Approach 1:
The patent employs prokaryotic expression systems (E. coli) instead of expensive mammalian cell lines, using a cost-effective biological platform that can be scaled up easily. The bacterial system produces the protein at a fraction of the cost of mammalian systems while maintaining protein stability through proper sequence design
Solution Approach 2:
The patent modifies the protein sequence by adding specific amino acids at the N-terminus (such as methionine, alanine, or serine) to prevent degradation. These sequence parameter changes stabilize the protein against proteolytic degradation while maintaining its biological activity, resolving the contradiction between protein stability and production cost
2Ease of manufacture
If recombinant human MYDGF is produced in bacterial expression systems with a C-terminal Histag, then the protein can be produced at lower cost, but the His-Tag poses a significant antigenicity risk when administered to human patients
Solution Approach 1:
The patent removes the C-terminal His-Tag from the protein sequence entirely, extracting the problematic element that causes antigenicity. Instead, it uses N-terminal amino acid modifications (methionine, alanine, or serine additions) that do not create antigenic epitopes, thereby eliminating the harmful effect while maintaining manufacturability
Solution Approach 2:
The patent applies local modifications only at the N-terminus of the protein sequence rather than adding tags throughout the protein. This localized modification approach stabilizes the protein against degradation without introducing widespread antigenic epitopes, resolving the contradiction between ease of manufacture and safety
3Productivity
If recombinant human MYDGF is expressed in heterologous systems, then the protein can be produced, but considerable degradation occurs with one or more amino acids at the N-terminus being degraded
Solution Approach 1:
The patent performs preliminary protective measures by adding specific amino acids (methionine, alanine, or serine) at the N-terminus before the protein is expressed in the heterologous system. These preemptive sequence modifications prevent proteolytic degradation during expression, ensuring high protein purity and preventing the formation of degradation products
4Quantity of substance
If larger-scale production of recombinant human MYDGF is attempted in mammalian cell systems, then therapeutic quantity can be produced, but the costs render the production unattractive from an economical perspective
Solution Approach 1:
The patent replaces expensive mammalian cell systems with inexpensive prokaryotic systems that can be scaled to produce therapeutic quantities. The bacterial expression system, when properly engineered with stabilizing N-terminal sequences, achieves cost-effective large-scale production while maintaining protein stability and activity
Solution Approach 2:
The patent changes the expression system parameter from mammalian to prokaryotic, fundamentally altering the production economy. This parameter change enables scalable production at lower costs while the N-terminal sequence modifications ensure protein stability, resolving the contradiction between quantity produced and production cost
Data Source
AI summary
The present invention generally relates to the field of recombinant gene expression in host cells. In particular, the invention relates to a recombinant human myeloid-derived growth factor (MYDGF) protein that exhibits a minimal degree of degradation upon expression in a host cell. The recombinant protein is therefore highly suitable for medical use, in particular for treating heart tissue damage and preventing cell death in myocardial tissue. The invention also provides a nucleic acid which encodes the recombinant protein and a host cell that expresses the recombinant protein. The invention also provides a method for producing the recombinant protein in a host cell.