Cysteine-Less Split Inteins for Reducing-Agent-Free Bioconjugation
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Solution Overview
Problem
Existing split-intein systems are limited by the presence of cysteine residues, which require reducing agents and can render mature proteins non-functional, making cysteine-less (CL) split-intein systems rare and difficult to obtain.
Innovation Solution
Engineered split-intein systems derived from Richelia sp., Pseudomonas aeruginosa 18, and Candidatus Brocadiales, with N-terminal and C-terminal sequences having high sequence identity, optionally including linkers and localization, affinity, or reporter tags, capable of catalyzing bioconjugation reactions at pH 6 to 8 and temperatures from 20°C to 50°C without reducing agents.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If cysteine-containing split-intein systems are used, then trans-splicing reactions can proceed, but reducing agents are required which can render mature proteins non-functional
Solution Approach 1:
The patent removes cysteine residues from the split-intein sequences through point mutations, extracting the problematic functional group that requires reducing agents. This creates cysteine-less split-intein systems that can perform trans-splicing without requiring reducing agents, thereby preserving mature protein functionality while maintaining the desired biochemical activity.
Solution Approach 2:
The patent modifies the chemical composition parameters of the intein sequences by substituting cysteine residues with other amino acids. This parameter change eliminates the need for reducing agents in the reaction system, allowing the trans-splicing to proceed under conditions that preserve disulfide bonds and maintain protein functionality.
2Reliability
If cysteine residues are removed to create cysteine-less split-inteins, then protein functionality is preserved, but such systems are rare and difficult to obtain
Solution Approach 1:
The patent divides the intein sequence into N-terminal and C-terminal halves, both of which are engineered to be cysteine-less. This segmentation allows the creation of functional split-intein systems without cysteine residues, expanding the range of compatible exteins while maintaining trans-splicing capability.
Solution Approach 2:
The patent systematically modifies the amino acid sequence parameters of the intein halves through point mutations to eliminate cysteine residues. This creates new cysteine-less split-intein variants that broaden extein compatibility while preserving the essential trans-splicing function.
3Reliability
If native split-intein sequences are used, then trans-splicing activity is achieved, but flexibility in extein choices is limited due to cysteine residue requirements
Solution Approach 1:
The patent extracts and removes the cysteine residues from native split-intein sequences, creating modified versions that eliminate the restriction on extein choices. This allows the trans-splicing system to be applied to a broader range of extein proteins without concerns about cysteine interference or reducing agent requirements.
Solution Approach 2:
The patent creates universal cysteine-less split-intein halves that can function with diverse extein partners. By removing the cysteine dependency, the system becomes universally applicable to various extein proteins, enhancing its multi-functionality and versatility across different protein engineering applications.
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 engineered systems facilitate robust and efficient bioconjugation reactions, tolerating a wide range of conditions, including various reducing agents, salts, and denaturing agents, while preserving extein functionality.
Implementation Method 1
The N-terminal and C-terminal split extein-intein halves recognize each other and catalyze protein trans-splicing reactions leading to mature proteins consisting of the N-terminal and C-terminal extein halves without intein polypeptides
Data Source
AI summary
Described in several example embodiments herein are engineered split intein polypeptides and systems thereof. Also described in several example embodiments, herein are methods of using the engineered split intein polypeptides and systems thereof, such as to catalyze a bioconjugation reaction.