Atypical Split Inteins Accelerating Protein Splicing Kinetics
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Solution Overview
Problem
Split inteins used in protein engineering face limitations such as slow kinetics, context-dependent efficiency, low expression levels, and suboptimal stability, which constrain their practical utility in protein purification and modification applications.
Innovation Solution
Development of atypical split inteins with accelerated splicing rates and enhanced activity under adverse conditions, specifically designed for N-terminal modification of proteins, utilizing short N-terminal fragments that can be easily synthesized and combined with other proteins or compounds through amide linkages.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional split inteins are used for protein modification, then the splicing reaction can occur, but the splicing rate is slow which limits productivity
Solution Approach 1:
The patent applies parameter changes by modifying the amino acid sequence of the split intein to create an optimized version with improved splicing kinetics. Specifically, the intein sequence was engineered to enhance the nucleophilic attack step and improve overall reaction rate, transforming the kinetic parameters of the splicing reaction to achieve faster processing while maintaining functionality.
2Reliability
If conventional split inteins are used under adverse conditions, then the splicing reaction can proceed, but the activity is suboptimal due to low stability
Solution Approach 1:
The patent improves stability under adverse conditions by engineering the intein sequence to enhance its structural robustness. The optimized intein maintains proper folding and catalytic activity across a broader range of temperatures and pH conditions, allowing reliable splicing reactions to proceed even in challenging cellular environments where conventional inteins would denature or become inactive.
3Productivity
If conventional split inteins are expressed in cells, then protein modification can be achieved, but expression levels are low which limits the amount of modified protein produced
Solution Approach 1:
The patent addresses low expression levels by optimizing the intein sequence for improved translational efficiency and protein stability. The engineered intein exhibits enhanced expression in heterologous systems, likely through modifications that improve ribosomal binding, reduce misfolding, or enhance cellular compatibility, thereby increasing the overall quantity of functional intein available for splicing reactions and subsequent protein modification.
4Productivity
If conventional split inteins are used for protein purification, then separation can be achieved, but the process is inefficient due to context-dependent efficiency
Solution Approach 1:
The patent enhances versatility by creating an optimized split intein that functions reliably across diverse protein contexts and experimental conditions. The engineered intein maintains consistent splicing efficiency regardless of the specific extein sequence or cellular environment, making it a universal tool applicable to various protein purification and modification protocols without requiring context-specific optimization.
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 new split inteins exhibit improved splicing rates and stability, enabling more efficient protein modifications and purifications, complementing existing methods like expressed protein ligation and transpeptidase-based strategies.
Implementation Method 1
An intein is an intervening protein domain that undergoes a posttranslational auto-processing event called protein splicing in which it excises itself from a host protein while tracelessly ligating its flanking polypeptide sequences (exteins) to form a native peptide bond
Implementation Method 2
Some exist naturally in split form, whereby each intein fragment is encoded on a separately expressed gene and must first associate prior to splicing in trans
Data Source
AI summary
The present disclosure relates to atypical split N- and C-inteins and variants thereof. This disclosure also relates to complexes comprising the split N- or C-inteins of this disclosure and a compound of interest and compositions comprising said complexes. In addition, this disclosure relates to methods of using the atypical split N- and C-inteins.