Evolved Sortase Enzyme for Aβ Labeling
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Solution Overview
Problem
Naturally occurring bond-forming enzymes like sortases have limited substrate specificity and efficiency, restricting their application in modifying proteins, especially for proteins lacking specific recognition sequences, and require genetic manipulation, which can be cumbersome and disrupt native protein structure and function.
Innovation Solution
Laboratory evolution of bacterial transpeptidase sortase A to recognize the LMVGG sequence, enabling site-specific protein modification without genetic manipulation, with a >1,400-fold change in substrate preference, allowing for efficient labeling and inhibition of Aβ aggregation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If naturally occurring sortases are used with their native substrate specificity, then the enzyme structure remains simple and easy to obtain, but the substrate spectrum is limited and cannot modify proteins lacking specific recognition sequences
Solution Approach 1:
The patent applies parameter changes by modifying the enzyme's substrate recognition parameters through directed evolution. Specifically, the sortase enzyme was evolved to recognize and bind to LMVGG sequences instead of the native LPXTG sequences, fundamentally changing the enzyme's substrate specificity parameter to expand its applicability to a broader range of proteins including Aβ.
Solution Approach 2:
The patent employs dynamics by creating an adaptable enzyme system that can evolve its substrate preference. The sortase enzyme was subjected to iterative mutagenesis and selection processes, allowing it to dynamically adjust its substrate recognition characteristics from LPXTG to LMVGG, thereby adapting to different protein targets without requiring structural redesign.
2Adaptability or versatility
If genetic manipulation is performed to add sortase recognition motifs to target proteins, then the protein can be modified by sortase, but the process becomes cumbersome and may disrupt native protein structure and function
Solution Approach 1:
The patent applies the inversion principle by reversing the traditional approach. Instead of modifying the target protein to include sortase recognition motifs (LPXTG at C-terminus or GGG at N-terminus), the invention inverts the strategy by evolving the sortase enzyme itself to recognize endogenous protein sequences like LMVGG in Aβ, thereby eliminating the need for genetic manipulation of the target protein.
Solution Approach 2:
The patent employs self-service by enabling the sortase enzyme to utilize naturally occurring sequences within the target protein as substrates. The evolved sortase recognizes and binds to LMVGG sequences that are already present in Aβ, allowing the enzyme to modify the protein without requiring external genetic engineering or addition of artificial recognition motifs.
3Productivity
If naturally occurring sortases are used, then the enzyme system is simple and well-characterized, but the reaction efficiency is low and cannot achieve high sensitivity labeling
Solution Approach 1:
The patent applies periodic action through iterative cycles of mutagenesis and selection. The sortase enzyme was subjected to multiple rounds of directed evolution, where each cycle involved introducing mutations, selecting for improved LMVGG recognition and catalytic activity, and repeating the process. This periodic iteration progressively enhanced the enzyme's reaction efficiency and sensitivity for Aβ labeling.
Solution Approach 2:
The patent employs feedback mechanisms in the directed evolution process, where the enzyme's performance on LMVGG substrates was continuously monitored and used to guide further mutagenesis. Selection pressures were applied based on measured catalytic activity and substrate binding affinity, allowing the enzyme to evolve high reaction efficiency through feedback-driven optimization of its active site and substrate recognition properties.
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 evolved sortase effectively labels endogenous Aβ in human cerebrospinal fluid with high sensitivity, inhibits Aβ aggregation, and modifies proteins without disrupting their native structure, expanding the scope of sortase applications in research, therapy, and diagnostics.
Implementation Method 1
sortases catalyze a transpeptidation reaction that results in the conjugation of a peptide comprising a C-terminal sortase recognition motif with a peptide comprising an N-terminal sortase recognition motif
Implementation Method 2
a sortase that binds substrates comprising the amino acid sequence LMVGG
Data Source
AI summary
Evolved sortases exhibiting enhanced reaction kinetics and/or altered substrate preferences are provided herein, for example evolved sortases that bind recognitions motifs comprising a LMVGG [SEQ ID NO: 3] sequence. Also provided are methods (e.g., orthogonal transpeptidation and diagnostics methods) for using such sortases.