Monomeric Ocr Fusion Tag for Protein Solubilization
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Solution Overview
Problem
Current fusion tags, such as MBP, face challenges during protein purification and processing, including incomplete binding to affinity resins and interference with downstream applications like NMR or crystallization, due to precipitation issues and difficulty in removing the tag from the passenger protein.
Innovation Solution
A monomeric Ocr protein variant with specific mutations (e.g., F53R and V77D) is used as a fusion tag, which prevents dimerization and has an acidic pI, enhancing solubility and serving as an effective affinity handle for purification, allowing for efficient cleavage and separation from the passenger protein.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If MBP fusion tag is used to enhance solubility, then solubility of passenger protein is improved, but binding to affinity resin becomes unreliable and tag removal becomes difficult
Solution Approach 1:
The patent modifies the MBP fusion tag by introducing specific amino acid substitutions (e.g., E100Q, E103Q, E106Q, E107Q, E109Q, E110Q, E112Q, E113Q, E115Q, E116Q) to alter its binding properties. These parameter changes in the tag sequence improve both solubility enhancement and affinity resin binding reliability, resolving the contradiction between solubility improvement and binding reliability.
2Stability of the object's composition
If MBP fusion tag is used during expression, then solubility is enhanced, but precipitation occurs after cleavage and tag removal interferes with downstream applications
Solution Approach 1:
The patent introduces specific amino acid substitutions in the MBP fusion tag sequence to modify its behavior after proteolytic cleavage. These parameter changes prevent precipitation events that normally occur after tag removal, ensuring the passenger protein remains soluble and suitable for downstream applications like NMR and crystallization.
3Ease of manufacture
If MBP fusion tag is used for purification, then affinity handling is provided, but incomplete removal of tag interferes with downstream applications
Solution Approach 1:
The patent modifies the MBP fusion tag sequence with specific amino acid substitutions that enhance the efficiency and completeness of proteolytic cleavage. These parameter changes ensure complete tag removal while maintaining ease of purification, eliminating interference with downstream applications such as NMR spectroscopy and protein crystallization.
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 monomeric Ocr protein variant effectively solubilizes and purifies proteins, reducing aggregation, facilitating high-throughput cloning and expression, and enabling efficient purification and downstream applications without the need for extensive tag removal.
Implementation Method 1
a monomeric Ocr protein variant with specific mutations (e.g., F53R and V77D) is used as a fusion tag, which prevents dimerization and has an acidic pI, enhancing solubility
Implementation Method 2
serving as an effective affinity handle for purification
Data Source
AI summary
The present invention relates to fusion proteins. In particular, the present invention relates to protein tags for use in protein solubilization and purification.