Orthogonal bdSUMO Cleavage Tags for Stable Eukaryotic Expression
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Solution Overview
Problem
Existing protein expression systems, particularly in eukaryotic hosts, face challenges in introducing eukaryotic post-translational modifications and proper folding, and current protease systems suffer from poor specificity, substrate turnover, and interference with target protein function, especially when using ubiquitin-like modifiers like SUMO.
Innovation Solution
Development of a fusion protein system comprising bdSUMO mutants and bdSENP1 protease variants with orthogonal specificity, allowing stable expression and efficient tag removal in eukaryotic systems, using an in vivo selection system in E. coli to evolve protease/PCS pairs.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If traditional proteases (thrombin, Factor Xa, TEV protease) are used for tag removal, then tag cleavage can be achieved, but specificity is poor leading to degradation of target protein
Solution Approach 1:
The invention changes the substrate recognition parameters by using SUMO proteases that recognize the 3D fold of SUMO proteins rather than short linear sequences. This parameter change in recognition mechanism eliminates off-target cleavage while maintaining efficient tag removal, resolving the contradiction between cleavage efficiency and protein degradation.
Solution Approach 2:
The SUMO protein acts as an intermediary between the protease and the target protein. The SUMO tag mediates the interaction by providing a specific 3D structure that the SUMO protease recognizes, enabling specific tag cleavage without affecting the target protein structure or function.
2Productivity
If SUMO proteases are used in eukaryotic hosts, then efficient tag cleavage is achieved, but endogenous SUMO proteases cause premature tag cleavage
Solution Approach 1:
The invention introduces local quality changes by mutating specific residues in the SUMO tag sequence that are critical for recognition by endogenous eukaryotic SUMO proteases. These localized mutations preserve the 3D fold necessary for SUMO protease activity while eliminating recognition by host proteases, achieving both high productivity and tag stability.
Solution Approach 2:
Instead of trying to protect the tag from all proteases, the invention inverts the approach by designing a tag that is specifically recognized only by the introduced SUMO protease variant while being invisible to endogenous proteases. This inversion of recognition specificity resolves the contradiction between needing efficient cleavage and maintaining stability.
3Ease of manufacture
If affinity tags are used to facilitate purification, then standardized purification can be achieved, but tags interfere with target protein function
Solution Approach 1:
The invention extracts the interfering tag from the target protein through site-specific proteolytic cleavage. The SUMO tag is removed completely after purification, leaving no residual sequences that could interfere with target protein function, while still providing the purification benefit during the manufacturing process.
Solution Approach 2:
The tag is used preliminarily during the purification process and then removed. The SUMO tag performs its facilitation function during manufacturing, and subsequent proteolytic cleavage removes it before the protein is used, eliminating any potential interference with target protein function in downstream applications.
4Adaptability or versatility
If proteases with broad specificity are used, then multiple PCS types can be cleaved, but orthogonality is lost preventing sequential purification
Solution Approach 1:
The invention segments the protease specificity by using different SUMO protease variants that each recognize distinct mutated SUMO tag sequences. This segmentation of recognition specificity enables orthogonal cleavage of different tags in sequential purification steps while maintaining the versatility to handle multiple protein complexes.
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 system enables efficient and orthogonal cleavage of bdSUMO fusion proteins, reducing toxicity and improving protein purification efficiency by using bdSUMO Mut1 and bdSENP1 MutB, which are resistant to wild-type SUMO proteases, thus facilitating stable expression and purification in eukaryotic hosts.
Implementation Method 1
bdSENP1 MutB, which are resistant to wild-type SUMO proteases, thus facilitating stable expression and purification in eukaryotic hosts
Implementation Method 2
The affinity capture and proteolytic release strategy is a particularly efficient implementation of affinity chromatography. A tag-PCS-target protein fusion is bound through the tag to an affinity matrix
Data Source
Figure 1
Figure 2A~2F
Figure 3A~3B
AI summary
The present invention relates to a fusion protein, comprising the structure N- PCSY - degSigN - M - PCSX - degSigC -C; wherein N represents the N-terminus; PCSY and PCSX each represent a protease cleavage site (PCS), which differ from each other in at least one amino acid residue; degSigN represents a degradation signal which promotes degradation of the fusion protein in a host cell if PCSY is cleaved by a protease such that the first amino acid of degSigN becomes the new N-terminus of the remaining fusion; M represents a cytoplasmic selection marker; and degSigC represents a second degradation signal which promotes degradation of the fusion protein in a host cell if PCSX is not cleaved by a protease; and C represents the C-terminus. Further provided is a nucleic acid construct, comprising a nucleic acid sequence coding for said fusion protein, a nucleic acid expression construct library, comprising a plurality of such nucleic acid expression constructs in diversified form, and methods using the fusion protein and nucleic acid constructs coding therefor. Finally, the present invention provides variants of bdSUMO and bdSENP1 which have been identified by the methods of the present disclosure, and which exhibit improved properties over existing orthogonal protease/protease cleavage site-pairs which are currently used with wild-type bdSUMO and wildtype bdSENP1.