Mutant Peptide Tag and Catcher Pair for Isopeptide Bond Formation
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Solution Overview
Problem
Current peptide tag and polypeptide binding partner systems, such as SpyTag/SpyCatcher, face limitations in reaction rate at cellular expression levels, necessitating the development of modified linkers with enhanced reaction rates while maintaining stability and utility.
Innovation Solution
The amino acid sequences of SpyTag and SpyCatcher peptides are modified to increase reaction rates by introducing specific mutations, particularly at the N- and C-terminus, resulting in a mutant peptide tag and polypeptide binding partner pair with improved reaction rates without compromising thermal stability or expression efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If peptide tag and polypeptide binding partner sequences are modified to increase reaction rates, then reaction rate is improved, but thermal stability or expression efficiency may be compromised
Solution Approach 1:
The patent applies parameter changes by systematically modifying amino acid sequences at specific positions (N- and C-terminus) to enhance reaction rate while monitoring and maintaining thermal stability. Multiple sequence variants are generated with controlled parameter changes to optimize the reaction rate without compromising stability.
Solution Approach 2:
The patent applies local quality by introducing mutations specifically at the N- and C-terminus regions of the peptide tag and polypeptide binding partner, rather than throughout the entire sequence. This localized modification approach allows enhancement of reaction rate at critical interfaces while preserving the thermal stability properties of the core structure.
2Productivity
If peptide tag and polypeptide binding partner sequences are modified to increase reaction rates, then reaction rate is improved, but expression efficiency may be compromised
Solution Approach 1:
The patent applies parameter changes by systematically modifying amino acid sequences at specific positions (N- and C-terminus) to enhance reaction rate while monitoring and maintaining thermal stability. Multiple sequence variants are generated with controlled parameter changes to optimize the reaction rate without compromising stability.
Solution Approach 2:
The patent applies local quality by introducing mutations specifically at the N- and C-terminus regions of the peptide tag and polypeptide binding partner, rather than throughout the entire sequence. This localized modification approach allows enhancement of reaction rate at critical interfaces while preserving the thermal stability properties of the core structure.
3Productivity
If specific mutations are introduced at N- and C-terminus, then reaction rate increases up to 10-fold, but sequence complexity increases
Solution Approach 1:
The patent applies local quality by introducing mutations specifically at the N- and C-terminus regions of the peptide tag and polypeptide binding partner, rather than throughout the entire sequence. This localized modification approach allows enhancement of reaction rate at critical interfaces while preserving the thermal stability properties of the core structure.
Solution Approach 2:
The patent applies segmentation by dividing the modification strategy into distinct regions (N-terminus and C-terminus) and treating them separately with specific mutation patterns. This segmented approach to sequence design allows systematic optimization of reaction rate while maintaining manageability of sequence complexity.
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 modified mutant tag and catcher pair exhibit a significantly increased reaction rate, up to 10-fold, facilitating efficient covalent bonding even at low concentrations and in the presence of large proteins or detergents, while maintaining stability across various conditions.
Implementation Method 1
proteins and peptide tags with enhanced rate of spontaneous isopeptide bond formation
Data Source
AI summary
The present invention relates to a two-part linker comprising a peptide tag (peptide) and a polypeptide (protein) that is capable of spontaneously forming an isopeptide bond, particularly wherein: a) said peptide comprises an amino acid sequence as set forth in SEQ ID NO: 1, wherein: (i) X at position 1 is arginine or no amino acid; (ii) X at position 2 is glycine or no amino acid; (iii) X at position 5 is histidine or threonine; (iv) X at position 11 is alanine, glycine or valine; and (v) X at position 14 is arginine or lysine, wherein when X at position 1 is no amino acid, X at position 2 is no amino acid; and b) said polypeptide comprises: i) an amino acid sequence as set forth in SEQ ID NO: 2; ii) a portion of (i) comprising an amino acid sequence as set forth in SEQ ID NO: 101; iii) an amino acid sequence with at least 80% sequence identity to a sequence as set forth in SEQ ID NO: 2, wherein said amino acid sequence comprises a lysine at position 34, a glutamic acid at position 80 and one or more of the following: 1) threonine at position 5; 2) proline at position 16; 3) arginine at position 40; 4) histidine at position 65; 5) proline at position 92; 6) aspartic acid at position 100: 7) glutamic acid at position 108; and 8) threonine at position 116, wherein the specified amino acid residues are at positions equivalent to the positions in SEQ ID NO: 2; or iv) a portion of (iii) comprising an amino acid sequence with at least 80% sequence identity to a sequence as set forth in SEQ ID NO: 101, wherein the amino acid sequence comprises a lysine at position 10, a glutamic acid at position 56 and one or more of the following: 1) arginine at position 16; 2) histidine at position 41; 3) proline at position 68; and 4) aspartic acid at position 76, wherein the specified amino acid residues are at positions equivalent to the positions in SEQ ID NO: 101, and wherein said peptide and polypeptide are capable of spontaneously forming an isopeptide bond between the aspartic acid residue at position 10 of SEQ ID NO: 1 and the lysine residue at position 34 of SEQ ID NO: 2 or position 10 of SEQ ID NO: 101.