Intein-Modified Enzyme Control via Self-Splicing
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Solution Overview
Problem
Proteins such as hydrolytic enzymes, when expressed in plants or microbial hosts, can cause undesirable phenotypic effects due to their hydrolytic activity, leading to degradation of plant components or other detrimental effects on growth and performance.
Innovation Solution
Development of isolated proteins or nucleic acids with specific amino acid sequences that have at least 90% identity to certain sequences, incorporating intein sequences for controlled activity, allowing for modification of enzyme activity through intein insertion and splicing, which can be induced by conditions like temperature changes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If hydrolytic enzymes (xylanases, cellulases) are expressed in plants or microbial hosts, then industrial and agricultural applications are enabled, but undesirable phenotypic effects occur due to hydrolytic activity degrading plant components or affecting growth
Solution Approach 1:
The enzyme protein is segmented into two functional parts: an intein self-splicing module and an extein catalytic domain. The intein segment can be removed through self-splicing, allowing the enzyme to be produced in a inactive form that does not harm the host, and then activated when needed. This segmentation resolves the contradiction by separating the harmful production phase from the useful functional phase.
Solution Approach 2:
The enzyme is produced in advance in an inactive form with the intein segment present, preventing harmful hydrolytic activity during host growth. The active catalytic domain is prepared but held in check by the intein until splicing occurs. This preliminary action allows safe expression in the host while maintaining the capability for future useful function.
Solution Approach 3:
The enzyme transitions from a static inactive state (with intein present) to a dynamic active state (after splicing). The self-splicing mechanism allows the enzyme to change its functional state based on environmental conditions or developmental timing, enabling harmful activity to be avoided during production while maintaining usefulness when activated.
2Power
If enzymes with hydrolytic activity are expressed, then catalytic function is achieved, but detrimental effects on host growth and physiological performance occur
Solution Approach 1:
The intein acts as an intermediary element between the catalytic domain and the host system. It mediates the relationship by being removable through self-splicing, allowing the catalytic domain to function without directly impacting host growth. The intein serves as a temporary placeholder that can be removed when the catalytic function is needed, protecting host reliability while enabling enzymatic power.
Solution Approach 2:
The enzyme's functional parameters change based on the presence or absence of the intein segment. When the intein is present, the enzyme has low or no catalytic activity toward plant components, ensuring host health. After self-splicing removes the intein, the catalytic parameters are activated, enabling useful hydrolytic function. This parameter change resolves the contradiction between power and reliability.
3Ease of operation
If intein sequences are inserted into enzymes to control activity, then predefined activity levels are achieved, but protein sequence complexity increases
Solution Approach 1:
The intein sequence performs self-service through self-splicing, automatically removing itself and activating the catalytic domain without requiring external intervention. This self-service mechanism simplifies the control system compared to using complex regulatory proteins or external enzymes, as the intein autonomously manages its own removal and the enzyme's activation, reducing overall system complexity while achieving predefined activity control.
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
Enables the expression of proteins with predefined activity levels, reducing detrimental effects on hosts and allowing for increased activity post-splicing, thereby improving the use of proteins like xylanases and cellulases in industrial and agricultural applications without interfering with host growth or performance.
Implementation Method 1
Intein sequences can be inserted into proteins at positions that do not interfere with the folded structure or function of the protein. The intein sequences can be removed from the protein through self-splicing.
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.