FKBP12 Fusion Polypeptide for Immunogenic Epitope Stabilization
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Solution Overview
Problem
Current methods for producing therapeutic antibodies face challenges in accessing and providing sufficient quantities of immunogenic peptides with specific secondary structures, particularly those that are difficult to stabilize or present in conventional immunization protocols.
Innovation Solution
The development of fusion polypeptides comprising fragments of peptidyl-prolyl cis/trans isomerase or FKBP family members, which are recombinantly produced and used for immunization to generate antibodies that specifically bind to hidden or buried epitopes, and for displaying peptide and secondary structures in methods like antibody screening and epitope mapping.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If conventional immunization protocols are used, then immunogens can be administered to generate antibodies, but sufficient quantities of immunogenic peptides with specific secondary structures cannot be obtained
Solution Approach 1:
The patent uses FKBP12 as an intermediary scaffold protein to which immunogenic peptides are conjugated. This mediator enables the presentation of specific secondary structures (alpha-helices, beta-sheets, turns) that cannot be adequately presented by conventional peptide immunogens alone, thereby resolving the contradiction between quantity and structural adaptability
Solution Approach 2:
The invention creates composite immunogens by combining FKBP12 scaffold protein with immunogenic peptide sequences. This composite structure provides both the stability and quantity of the protein scaffold and the specific secondary structure presentation capability, overcoming the limitations of using either component alone
2Quantity of substance
If recombinant polypeptides are produced in prokaryotic or eukaryotic cells, then sufficient quantities can be obtained, but the polypeptides may form insoluble inclusion bodies requiring solubilization
Solution Approach 1:
The patent employs E. coli as a disposable, inexpensive expression system for producing the FKBP12-immunogen fusion polypeptide. The system is designed for high-yield production where the expression host is replaced rather than maintained, simplifying the overall manufacturing process despite the challenge of inclusion body formation
Solution Approach 2:
The invention changes the physical-chemical parameters of the recombinant expression system by using mild solubilization conditions (non-denaturing detergents, chaotropic agents) and optimizing lysis conditions to maintain the stability of the FKBP12-immunogen fusion while solubilizing inclusion bodies, thereby reducing purification complexity
3Ease of manufacture
If immunogens are produced in soluble form, then purification is simpler, but specific secondary structures may not be stabilized
Solution Approach 1:
The FKBP12 scaffold acts as an intermediary that stabilizes the immunogenic peptide in a soluble state while maintaining its secondary structure. The scaffold's natural stability and solubility properties allow the conjugated immunogen to remain soluble and purifiable without losing its structural integrity
Solution Approach 2:
The invention segments the immunogen into two functional parts: the FKBP12 scaffold that provides solubility and stability, and the immunogenic peptide that provides the specific secondary structure. This segmentation allows each component to fulfill its optimal function while working together
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
These fusion polypeptides enable the generation of antibodies that target internal epitopes and provide stable, soluble, and monomeric presentations of immunogenic sequences, overcoming the limitations of conventional immunization by stabilizing the structure and increasing molecular weight for effective immunization and analysis.
Implementation Method 1
The fusion polypeptides are stable, soluble, and monomeric presentations of immunogenic sequences
Implementation Method 2
stabilizing the structure of the immunogenic polypeptide
Implementation Method 3
fragments of one or more peptidyl-prolyl cis/trans isomerase or FKBP family members
Data Source
AI summary
Herein is reported a fusion polypeptide according to formula INH2—S2—X1—S1—COOH (formula I)whereinX1 comprises either a random amino acid sequence or an amino acid sequence derived from a first polypeptide,S2 and S1 are non-overlapping amino acid sequences derived from a second polypeptide, and— denotes a peptide bond,wherein the second polypeptide is a polypeptide with peptidyl-prolyl cis/trans-isomerase activity (PPIase activity) or is derived from the FKBP-fold domain family, wherein X1 is inserted in place of the insert-in-flap-domain of the second polypeptide.