SARS-CoV 3CLpro Mutant for Precise Fusion Protein Cleavage
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Solution Overview
Problem
Fusion protein technology faces challenges in precisely cleaving protein tags from fusion proteins, leading to incomplete recovery of structurally intact target proteins due to imprecise cleavage.
Innovation Solution
A SARS-CoV 3CLpro mutant with a G substitution at position 25 is used to recognize and cleave a specific substrate site, allowing for precise release of target proteins from fusion proteins by recognizing the cleavage site P4P3P2QP1', where P4 and P3 are A, V, G, L, or I, P2 is L, V, or F, and P1' is G, A, V, L, I, S, T, M, C, D, E, Q, N, W, or F.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If a protein tag is used in fusion protein technology, then water solubility and purification are improved, but the structural and functional properties of the target protein are interfered with
Solution Approach 1:
The invention extracts the protein tag from the fusion protein through site-specific cleavage at the P4P3P2QP1' sequence. The mutated SARS-CoV 3CLpro protease specifically recognizes and cleaves between Q and P1' residues, removing the protein tag while preserving the target protein's structural integrity and functional properties.
Solution Approach 2:
The invention introduces a specific cleavage site sequence (P4P3P2QP1') as an intermediary between the protein tag and target protein. This sequence serves as a recognition site for the mutated protease, enabling precise separation of the tag from the target protein without affecting the target protein's structure or function.
2Productivity
If chemical or enzymatical cleavage is used to remove the protein tag, then the target protein is released, but imprecise cleavage results in failure to recover a structurally intact target protein
Solution Approach 1:
The invention changes the substrate specificity parameter of the SARS-CoV 3CLpro protease through site-directed mutagenesis (T25G mutation). This parameter change enables the protease to specifically recognize the P4P3P2QP1' sequence with high precision, cleaving between Q and P1' residues while maintaining the structural integrity of the target protein.
Solution Approach 2:
The invention introduces a specific local sequence (P4P3P2QP1') at the junction between the protein tag and target protein. This local sequence has unique properties that serve as a specific recognition site for the mutated protease, enabling precise cleavage at this specific location without affecting other regions of the target protein.
3Power
If the wild-type SARS-CoV 3CLpro is used for cleavage, then protease activity is maintained, but substrate specificity for the P4P3P2QP1' site is insufficient
Solution Approach 1:
The invention changes the substrate specificity parameter of the SARS-CoV 3CLpro protease through site-directed mutagenesis (T25G mutation). This single amino acid substitution at position 25 fundamentally alters the protease's substrate recognition properties, enabling it to specifically recognize and cleave the P4P3P2QP1' sequence with high precision while maintaining catalytic activity.
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 SARS-CoV 3CLpro mutant achieves precise cleavage between the Q and P1' residues, enhancing the recovery of structurally intact target proteins with a catalytic efficiency 43.5-fold higher than the wild-type protease, effectively addressing the imprecision in existing cleavage methods.
Implementation Method 1
The SARS-CoV 3CLpro mutant recognizes the cleavage site P4P3P2QP1′, in which each of P4 and P3, independently, is A, V, G, L, or I, P2 is L, V, or F, and P1′ is G, A, V, L, I, S, T, M, C, D, E, Q, N, W, Y, or F, and cleaves between Q and P1′
Data Source
AI summary
A mutated severe acute respiratory syndrome-associated coronavirus 3C-like protease and use thereof for cleaving a protein that includes a cleavage site recognizable by the mutated protease to yield a polypeptide fragment of interest.