Bis-met Histone N-terminal Extension for Tag-free Purification
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Solution Overview
Problem
Current methods for producing recombinant eukaryotic proteins, such as histones, face challenges in simplifying production and detection, particularly due to the use of affinity tags which can interfere with protein function and require subsequent removal for therapeutic applications.
Innovation Solution
A nucleic acid molecule encoding a polypeptide with two methionine residues as the first and second N-terminal amino acid residues linked via a peptide bond to a mature eukaryotic histone, allowing for improved detection and purification without the need for affinity tags, as these bis-met histones retain biological activity and are easily distinguishable from endogenous histones.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If affinity tags are used for purification and detection of recombinant proteins, then purification efficiency and detection ease are improved, but protein function may be interfered with and tag removal is required for therapeutic applications
Solution Approach 1:
The invention extracts only the essential detection function from affinity tags by using a minimal N-terminal methionine extension (Met1-Met2-His3) that can be easily removed. This allows purification and detection without the need for large affinity tags that interfere with protein function, as the minimal extension can be cleaved off to restore full protein activity for therapeutic applications
Solution Approach 2:
The invention applies local quality by placing the histidine tag only at the N-terminus with a specific minimal structure (Met1-Met2-His3) rather than using extensive affinity tags throughout the protein. This localized minimal tag provides sufficient purification and detection capability while minimizing interference with protein function, and can be easily removed if needed for therapeutic use
2Ease of operation
If large affinity tags are added to recombinant proteins, then purification and detection become easier, but translation, folding and activity are adversely affected
Solution Approach 1:
The invention uses a minimal N-terminal extension (Met1-Met2-His3) that serves as a temporary, disposable tag for purification and detection purposes. This short-lived minimal tag provides the necessary functionality during production but can be easily removed afterward, avoiding the persistent interference that large affinity tags cause to protein translation, folding and activity
3Device complexity
If affinity tags are used for recombinant protein production, then purification is simplified, but subsequent tag removal is necessary for therapeutic applications
Solution Approach 1:
The invention extracts only the minimal necessary components for purification (N-terminal Met1-Met2-His3) rather than using extensive affinity tags. This minimal extension can be easily removed by standard proteases, significantly reducing the time and complexity of tag removal compared to large affinity tags, while still providing simplified purification during production
Solution Approach 2:
The invention changes the size parameter of the affinity tag from large (traditional His-tag, GST, etc.) to minimal (Met1-Met2-His3, approximately 3 amino acids). This parameter change maintains the purification functionality while dramatically reducing the time and complexity required for subsequent tag removal, making the process feasible for therapeutic applications
Data Source
AI summary
The present invention provides a nucleic acid molecule which encodes a polypeptide consisting of two methionine residues as the first and second N-terminal amino acid residues linked via a peptide bond to a mature eukaryotic histone. The present invention furthermore relates to a vector containing said nucleic acid molecule, a host transformed with said vector, polypeptides encoded by the nucleic acid molecule and pharmaceutical and diagnostic compositions. The present invention also relates to the use of the nucleic acid molecule, vectors, hosts and the polypeptide of the invention for the preparation of a composition for the treatment of diseases. Furthermore, the present invention relates to a method of testing for the presence of the nucleic acid molecule or the polypeptide in a sample and to a kit.