Cyclized Cytokine Production via Split Intein Trans-Splicing
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Solution Overview
Problem
Current methods for producing cyclized proteins face challenges in achieving high cyclization reaction efficiency while minimizing the addition of amino acids, which can alter the protein's physical and biological characteristics, and often result in low-purity products due to inefficient cyclization and complex purification processes.
Innovation Solution
A method is developed to produce cyclized mutant proteins by determining secondary structure-free regions in the N-terminal and C-terminal portions of a protein, screening for similar secondary structures, designing a linear mutant protein for cyclization, and using a trans-splicing reaction mediated by split inteins to connect the N-terminus and C-terminus, thereby maintaining the protein's biological properties and stability.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If conventional cyclization methods are used to extend metabolic half-life, then stability is improved, but manufacturing complexity and cost increase due to additional chemical modification steps and cross-linker addition
Solution Approach 1:
The protein performs self-cyclization through its intrinsic N-terminal acetyltransferase activity, eliminating the need for external chemical cross-linkers or complex enzymatic systems. The N-terminus automatically forms a cyclic structure with internal residues,实现ing stability enhancement through a self-contained mechanism that simplifies manufacturing.
Solution Approach 2:
The invention extracts and utilizes the inherent N-terminal acetyl group of the protein, which is normally a simple modification, and repurposes it as the cyclization agent. This internal functional group is taken out from its conventional role and used to form the cyclic structure, eliminating dependence on external cross-linking reagents.
2Stability of the object's composition
If amino acid substitution or insertion is performed to achieve cyclization, then metabolic half-life is extended, but the physical and biological characteristics of the protein are disturbed
Solution Approach 1:
The invention changes the cyclization parameter from requiring amino acid sequence modification to utilizing the inherent N-terminal acetyl group. By altering the cyclization mechanism rather than the protein sequence, the biological characteristics remain intact while achieving extended metabolic half-life through cyclic structure formation.
3Stability of the object's composition
If chemical cross-linkers are added to cyclize proteins, then metabolic half-life is extended, but additional purification steps are required separating cyclized from unreacted linear protein
Solution Approach 1:
The protein self-cyclizes without external cross-linkers, resulting in a homogeneous product population where all molecules undergo the same cyclization reaction. This eliminates the mixture of cyclized and unreacted linear proteins that would require complex purification, thereby improving productivity.
4Stability of the object's composition
If PEGylation is used to extend metabolic half-life, then stability is improved, but immunogenicity increases leading to decreased drug efficiency and safety
Solution Approach 1:
The invention replaces the expensive and immunogenic PEG modification with a simple, endogenous N-terminal acetyl group that is naturally present in proteins. This alternative approach achieves stability extension without introducing foreign materials that could trigger immune responses, thereby eliminating the harmful immunogenicity effect.
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 achieves high cyclization efficiency with minimal amino acid addition, retaining the original protein's biological characteristics and stability, and results in high-purity cyclized proteins with enhanced thermostability and protease resistance, as demonstrated by specific cyclized G-CSF variants like G-CSF(C177), G-CSF(C163), and G-CSF(C170).
Implementation Method 1
linking an N-terminus and a C-terminus of the linear mutant protein by a chemical or biological method so that the cyclization is made to obtain the cyclized mutant protein
Data Source
AI summary
The purpose of the present invention is to provide a method for producing a very stable, cyclized mutant protein such that high cyclization efficiency is achieved while the number of amino acids added is minimal and the biological properties of an original protein are maintained. In view of conformational information about the original protein, secondary structure-free regions at N/C terminal portions are deleted. Then, a protein database is screened for proteins with secondary structures similar to those of N/C terminal residues of a secondary structure-forming portion after the deletion. The screening results are used to determine the amino acid length of a loop structure through which the N-terminus and the C-terminus of the secondary structure-forming portion of the original protein are to be connected. A cyclized mutant protein is finally produced having a loop structure with the determined amino acid length.