Truncated DME Polypeptide for Improved Solubility and Yield
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Solution Overview
Problem
Current methods for producing DEMETER (DME) protein in E. coli face challenges due to its large size and poor solubility, limiting expression yield and stability, which hampers the efficient excision of 5-methylcytosine from DNA.
Innovation Solution
Engineering DME by removing the interdomain region 1 (IDR1) and replacing it with a short linker peptide, resulting in a truncated form (DMEΔN677ΔIDR1::lnk) that is more soluble and stable, allowing for higher expression levels and improved purification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If full-length DME protein is produced in E. coli, then demethylase activity is maintained, but expression yield and solubility are poor
Solution Approach 1:
The patent extracts and removes the problematic interdomain region 1 (IDR1) from the full-length DME protein. This deletion resolves the solubility and expression issues while preserving the essential demethylase activity located in other domains of the protein.
Solution Approach 2:
The patent changes the structural parameters of the DME protein by deleting specific amino acid sequences (IDR1 region). This structural modification transforms the protein from insoluble/full-length to soluble/truncated form, improving expression yield while maintaining functional activity.
2Reliability
If full-length DME protein is produced in E. coli, then demethylase activity is maintained, but protein stability is poor
Solution Approach 1:
The unstable IDR1 interdomain region is extracted and removed from the protein structure. This elimination of the unstable region directly improves overall protein stability while the remaining stable domains maintain demethylase function.
3Reliability
If full-length DME protein is produced in E. coli, then complete functional domains are present, but purification is difficult
Solution Approach 1:
By removing the IDR1 region that complicates purification, the truncated protein structure becomes easier to manufacture and purify. The essential functional domains remain intact, ensuring complete demethylase 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 truncated DMEΔN677ΔIDR1::lnk form maintains demethylase activity, significantly increasing expression yield and solubility, and is successfully purified with enhanced stability, facilitating efficient 5-methylcytosine excision from DNA.
Implementation Method 1
the polypeptide excises methylated cytosines in DNA when contacted to DNA comprising methylated cytosines
Data Source
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AI summary
Improved active DEMETER polypeptides with internal deletions are provided.