Class C Sortase Variants for Isopeptide Protein Labeling
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Solution Overview
Problem
Current bioconjugation methods, such as those using Staphylococcus aureus Sortase A (Sa-SrtA), are limited in their ability to modify proteins at sites other than the N- and C-termini and are susceptible to proteolysis, as they primarily form peptide bonds which are not as stable as isopeptide bonds.
Innovation Solution
Development of bacterial Class C sortase variants with specific mutations in the lid region, like Corynebacterium diphtheriae sortase (Cd-SrtA), which can form threonine-lysine isopeptide bonds, enabling site-specific modification of proteins across their surface and providing stability against proteolysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If conventional Sa-SrtA is used for protein modification, then the method is simple and well-established, but the modification is limited to N- and C-termini and the peptide bonds are susceptible to proteolysis
Solution Approach 1:
The patent changes the chemical parameters of the bond formation by using isopeptide bonds instead of conventional peptide bonds. This chemical parameter change enables modification at surface sites (improving versatility) while simultaneously achieving greater stability against proteolysis (improving reliability), thus resolving the technical contradiction.
Solution Approach 2:
The invention creates a composite conjugation system that combines isopeptide bond formation with conventional peptide bond formation. This allows proteins to be modified at multiple sites with different bond types, achieving both expanded versatility and enhanced stability in the same protein modification system.
2Adaptability or versatility
If conventional Sa-SrtA is used, then the reagent is simple and widely adopted, but it cannot modify surface sites of proteins
Solution Approach 1:
By changing the catalytic parameters of the sortase enzyme to accept surface lysine residues as substrates, the patent enables surface site modification without fundamentally redesigning the reagent architecture. This parameter change achieves new functionality while maintaining relative simplicity.
Solution Approach 2:
The engineered sortase reagent is designed to perform multiple functions: it can still form conventional peptide bonds at N- and C-termini while also forming isopeptide bonds at surface lysine sites. This multi-functionality expands versatility without requiring entirely separate reagents for different modification types.
3Reliability
If isopeptide bonds are formed using the new sortase variants, then bond stability and site-specific modification are improved, but the reagent complexity increases
Solution Approach 1:
The patent introduces specific amino acid mutations (parameter changes) in the lid region of the sortase enzyme to enable isopeptide bond formation. These targeted parameter changes achieve the desired stability improvement while keeping the overall reagent structure relatively simple and manageable.
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 enzymatic reagents allow for the formation of isopeptide bonds, enabling labeling of new sites on proteins and forming stable linkages that are less susceptible to proteolysis, and can be used in conjunction with conventional Sa-SrtA for multiple molecule attachment to a single protein, enhancing the versatility and stability of protein modifications.
Implementation Method 1
The new enzyme reagents comprise bacterial Class C sortase variant polypeptides having one or more amino acid mutations in the lid region of the sortases, mutations that unexpectedly confer the enzymes with an ability to catalyze threonine-lysine isopeptide bond formation in vitro
Implementation Method 2
The new enzymatic reagents disclosed herein can create isopeptide bonds, not peptide bonds
Data Source
AI summary
Covalently cross-linked pilus polymers displayed on the cell surface of Gram-positive bacteria are assembled by class C sortase enzymes. These pilus-specific transpeptidases located on the bacterial membrane catalyze a two-step protein ligation reaction—first, cleaving the LPXTG motif of one pilin protomer to form an acyl-enzyme intermediate, and second, joining the terminal threonine to the nucleophilic lysine residue residing within the pilin motif of another pilin protomer. Informed by the high-resolution crystal structures of corynebacterial pilus-specific sortase (SrtA) and by developing structural variants of the sortase enzyme whose catalytic pocket has been unmasked by activating mutations, we have developed new reagents capable of forming isopeptide bonds in vitro. The reagents disclosed herein can catalyze ligation of isolated SpaA domains in vitro provide a facile and versatile new platform for protein engineering and bio-conjugation that has major implications for biotechnology.