Split Intein Transmembrane Domain Reconstitution
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Solution Overview
Problem
Current methods for biophysical characterization of transmembrane proteins rely on denaturing detergents that interfere with protein structure and function, making efficient expression and reconstitution challenging.
Innovation Solution
A transmembrane domain is covalently bound to a first intein of a split intein pair and embedded within a phospholipid layer, allowing for the synthesis of a fusion protein through split intein-mediated ligation without denaturing conditions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If denaturing detergents are used for transmembrane protein reconstitution, then the reconstitution process becomes feasible, but the protein structure and function are interfered with
Solution Approach 1:
The invention extracts and removes the harmful denaturing detergents from the reconstitution process by using a split intein system that enables detergent-free reconstitution. The transmembrane domain is reconstituted into phospholipid bilayers without requiring denaturing conditions, thereby preserving protein structure and function while maintaining reconstitution feasibility.
Solution Approach 2:
The split intein system acts as an intermediary mechanism that facilitates the reconstitution of transmembrane proteins without denaturing detergents. The intein-mediated ligation system enables the coupling of transmembrane domains to soluble proteins in a detergent-free environment, serving as a bridge between the transmembrane domain and the reconstitution process.
2Quantity of substance
If traditional overexpression and isolation methods are used, then transmembrane proteins can be obtained, but the process becomes tedious and requires denaturing conditions
Solution Approach 1:
The invention performs preliminary action by fusing the transmembrane domain to an intein moiety before reconstitution. This pre-fusion strategy eliminates the need for tedious overexpression and isolation steps, as the transmembrane domain is already prepared in a reconstitution-competent form with the intein tag attached, enabling direct incorporation into phospholipid bilayers.
Solution Approach 2:
The split intein system enables self-service by allowing the transmembrane domain to autonomously ligate to the soluble protein partner through intein-mediated trans-splicing after co-reconstitution. This self-ligation capability eliminates the need for external enzymatic ligation steps or complex purification procedures, significantly reducing the time and complexity of the overall process.
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
This method enables the efficient and functional reconstitution of transmembrane proteins within phospholipid layers, preserving their native structure and function, thereby overcoming the limitations of existing techniques.
Implementation Method 1
allowing for the synthesis of a fusion protein through split intein-mediated ligation
Data Source
AI summary
Provided herein, inter alia, are compositions and methods including transmembrane domains comprising a split intein and vesicles including transmembrane domains with a split intein. In embodiments, methods for generating vesicle embedded proteins in vitro without the use of denaturing agents, and their compositions, are provided.