Intein-Modified Enzyme Activity Control via Site-Specific Insertion
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Solution Overview
Problem
Proteins like xylanases and cellulases, when expressed in plants or microbial hosts, can cause undesirable phenotypic effects due to their hydrolytic activities, leading to degradation of plant components and other growth-related issues.
Innovation Solution
Development of intein-modified proteins by inserting inteins at specific sites within the target proteins, such as immediately prior to cysteine, threonine, or serine residues, or within 10 angstroms from the active site, to control protein activity and minimize detrimental effects, using methods like support vector machine analysis for site selection and expression constructs in transgenic plants.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If hydrolytic enzymes (xylanases, cellulases) are expressed in plants or microbial hosts, then industrial and agricultural applications are enabled, but undesirable phenotypic effects occur including degradation of plant components and detrimental effects on growth and physiological performance
Solution Approach 1:
An intein is inserted into the target protein sequence as an intermediary element. The intein acts as a controllable switch that can be activated or deactivated independently of the target enzyme expression. When the intein is in the 'off' state, it allows the target enzyme to function normally; when activated, it disrupts the target protein function, thereby controlling the harmful effects of the enzyme on host growth while maintaining productivity.
Solution Approach 2:
The invention changes the functional state of the target protein by modifying its primary structure through intein insertion. This structural modification allows the same protein to exhibit different functional parameters - either maintaining full enzymatic activity or having activity suppressed - depending on the intein's state, thus enabling control over the harmful effects without changing enzyme expression levels.
2Reliability
If intein is inserted into the target protein, then protein activity can be controlled to reduce harmful effects, but protein structure and function may be altered
Solution Approach 1:
The intein is inserted at specific local positions within the target protein sequence rather than uniformly throughout. By selecting insertion sites in regions less critical for catalytic function or structural integrity, the modification achieves reliable activity control while minimizing disruption to the overall protein structure and function.
Solution Approach 2:
The intein is designed to be a self-contained functional module that can be copied and inserted into different target proteins without requiring modification of the target protein's core functional domains. This modular approach allows control mechanisms to be replicated across different enzyme systems while preserving each target protein's native structure and function.
Data Source
AI summary
A method of predicting an intein insertion site in a protein that will lead to a switching phenotype is provided. The method includes identifying a plurality of C/T/S sites within the protein; selecting from the plurality of C/T/S/sites those that are ranked 0.75 or higher by a support vector machine, within ten angstroms of the active site of the protein, and at or near a loop-β-sheet junction or a loop-α-helix junction. A method of controlling protein activity and hosts including proteins with controlled activity are also provided. Also, intein modified proteins and plants containing intein modified proteins are provided.