Endostatin Mutants with ATP Binding Site Modifications
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for evaluating the biological activity of endostatin, a key anti-tumor therapeutic, are complex, subjective, and have low accuracy and reproducibility, and existing modifications to extend its half-life often reduce its biological activity.
Innovation Solution
Detection of ATPase activity in endostatin and its variants using biochemical assays, such as malachite green phosphate assay and ATP bioluminescence assay, to determine conformation and biological activity, along with genetic engineering to introduce mutations in the ATP binding motif to reduce ATPase activity and enhance anti-angiogenesis activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Duration of action of stationary object
If multiple sites PEG modifications are implemented on the amino of lysine side chain to prolong the half-life of ES, then the half-life is extended, but the biological activities are apparently reduced
Solution Approach 1:
The patent applies local quality by modifying only the N-terminal amino group of endostatin with a single PEG molecule, rather than modifying multiple lysine side chains throughout the protein. This localized modification approach preserves the biological activity of the protein while still achieving the desired extension of half-life, resolving the contradiction between prolonged duration and maintained reliability of biological function.
2Reliability
If single site PEG modification on the N-terminal is performed to enhance stability, then the stability and biological activities are improved, but the half-life extension is limited compared to multiple sites modification
Solution Approach 1:
The patent employs parameter changes by systematically varying the size and composition of the PEG molecule attached to the N-terminal of endostatin. By optimizing these parameters, the invention achieves an optimal balance between half-life extension and preservation of biological activity, demonstrating that careful parameter selection can resolve the contradiction between duration extension and activity maintenance.
3Ease of manufacture
If yeast is used to produce ES, then the protein is resolvable, but the N-terminal is truncated and the stability and biological activity are reduced
Solution Approach 1:
The patent applies the taking out principle by extracting the N-terminal region of endostatin that is prone to truncation during yeast expression, and replacing it with an artificial N-terminal sequence. This extracted and replaced segment prevents truncation issues while maintaining the resolvability advantage of yeast expression, thereby resolving the contradiction between ease of manufacture and reliability of the final product.
4Reliability
If the integrity of N-terminal is maintained to ensure stability and biological activity, then the activity is preserved, but the expression in prokaryotic system becomes difficult due to polar body formation
Solution Approach 1:
The patent introduces an intermediary element - an artificial N-terminal sequence with specific properties that act as a mediator between the prokaryotic expression system and the full-length endostatin protein. This intermediary sequence prevents polar body formation during prokaryotic expression while maintaining the integrity and functionality of the N-terminal region, thus resolving the contradiction between ease of manufacture and reliability of the final product.
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
This approach provides a sensitive, precise, and reliable method for evaluating biological activity and quality of endostatin, leading to the development of mutants with reduced ATPase activity but increased inhibitory effects on endothelial cell migration and tumor growth.
Implementation Method 1
ATP bioluminescence assay
Data Source
AI summary
The present invention discloses a new anti-tumor medicament comprising a mutant of endostatin. The mutant comprises a mutation in the ATP-binding site of endostatin and has a decreased ATPase activity and an increased anti-angiogenesis activity.