Segmented Atg4B Protease for Efficient Tag Removal
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Solution Overview
Problem
Current protease systems for tag removal in protein biochemistry face limitations such as low specific activity, limited specificity, and stability issues in eukaryotic hosts, particularly for UBL-specific proteases like Atg4, which are prone to cross-reactivity with endogenous enzymes.
Innovation Solution
The development of a Xenopus laevis Atg4B protease fragment (xIAtg4B 14-384) with enhanced substrate interaction, cleavage efficiency, and thermal stability, capable of cleaving Atg8-like proteins like xILC3B and xIGATE16, and its application in both prokaryotic and eukaryotic expression systems for efficient tag removal and protein purification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional protease systems (e.g., TEV protease, SUMO proteases) are used for tag removal, then tag removal function is achieved, but specific activity is low and productivity is limited
Solution Approach 1:
The Atg4B protease was divided into two functional domains: the catalytic domain (residues 1-384) was separated from the C-terminal extension (residues 385-478). The catalytic domain alone retains full proteolytic activity while improving stability and reducing cross-reactivity with endogenous enzymes in eukaryotic hosts, thereby resolving the contradiction between productivity and reliability.
Solution Approach 2:
The protease system was optimized by modifying the recognition site sequence from the native Atg8 FG/YG motif to an engineered PRS (Protease Recognition Site) with enhanced specificity. This parameter change in the substrate recognition sequence dramatically increased specific activity and prevented unwanted cleavage by endogenous proteases, improving both productivity and reliability simultaneously.
2Reliability
If full-length Atg4B protease is used, then substrate recognition capability is maintained, but thermal stability is reduced and device complexity increases
Solution Approach 1:
The full-length Atg4B protease (478 residues) was segmented into a catalytic domain (1-384 residues) and a C-terminal extension (385-478 residues). Structural analysis showed the C-terminal extension folds back onto the substrate-binding pocket, potentially interfering with substrate access. Removing this extension improved thermal stability while the catalytic domain maintained substrate recognition through the engineered PRS, resolving the contradiction between recognition accuracy and thermal stability.
3Adaptability or versatility
If UBL-specific proteases (Atg4, SUMO proteases) are used for tag removal, then specificity is improved, but cross-reactivity with endogenous enzymes occurs and reliability decreases
Solution Approach 1:
The natural Atg8 recognition motif (FG or YG at C-terminus) was modified to create an engineered Protease Recognition Site (PRS) with altered amino acid sequence. This parameter change in the recognition motif provides high substrate specificity for the catalytic domain of Atg4B while the modified sequence is not recognized by endogenous proteases, eliminating cross-reactivity and improving reliability without sacrificing versatility.
4Productivity
If existing protease systems are used, then tag removal is achieved, but manufacturing precision and purity of recombinant proteins are limited
Solution Approach 1:
The engineered PRS was designed with optimal spacing and amino acid composition between the affinity tag and the target protein. This parameter optimization ensures complete and precise cleavage at the PRS site, preventing incomplete tag removal or unwanted cleavage at other sites, thereby improving manufacturing precision and purification quality while maintaining high productivity through the active catalytic domain.
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
xIAtg4B 14-384 demonstrates robust substrate interaction, high thermal stability, and salt tolerance, achieving up to 30-50-fold higher activity than TEV protease at low temperatures, and allows for efficient purification of recombinant proteins with improved solubility and stability in eukaryotic hosts, overcoming the limitations of existing protease systems.
Implementation Method 1
The human Atg4B protease also contains a flexible extension at the C-terminus... xIAtg4B 14-384 demonstrates robust substrate interaction, high thermal stability, and salt tolerance, achieving up to 30-50-fold higher activity than TEV protease at low temperatures
Data Source
Figure 1A~1E
Figure 2A~2B
Figure 3A~3C
AI summary
The present invention belongs to the field of biotechnology. More specifically, the present invention provides a protease, a non-naturally occurring fusion protein comprising a corresponding protease recognition site, expression vectors encoding same, host cells comprising said expression vectors, kit of parts as well as methods applying the protease, fusion protein, and uses thereof, as defined in the claims. The presently disclosed protease/protease recognition site is particularly useful in methods requiring an orthogonal set of proteases, and is suitable for use in both prokaryotic and selected eukaryotic expression systems.