Polyepitopic Protein Amplification via SapI and SmaI Endonuclease Ligation
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Solution Overview
Problem
Existing methods for producing polyepitopic proteins and higher order polyepitope structures are limited by the inability to easily repeat DNA sequence amplification, leading to difficulties in achieving the desired number of repeating DNA segments, which hampers the production of improved vaccines and other applications.
Innovation Solution
A DNA vector system utilizing convergent DNA sequences recognized by Sapl endonuclease and Smal endonuclease, with an amplifying module and expression vector components, allows for directional ligation and autoligation to efficiently amplify and express polyepitopic proteins, enabling the production of proteins with arbitrary length and increased efficacy.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If conventional methods (IIS endonuclease SfaNI, BspM1, BbsI) are used for DNA sequence amplification, then some copying capability is achieved, but the ability to repeat amplification cycles and maintain ORF continuity deteriorates
Solution Approach 1:
The DNA sequence is divided into monomer units that can be independently amplified and then assembled through controlled ligation. The vector is segmented into specific modules (amplifying module with SapI sites, expression module, selection markers) that enable systematic repetition while maintaining integrity of each segment and the overall ORF.
Solution Approach 2:
The SapI restriction site serves as an intermediary element that facilitates controlled ligation between monomer units. This intermediary sequence enables reliable joining of DNA fragments while maintaining ORF continuity, acting as a bridge that ensures proper reading frame preservation during amplification cycles.
2Quantity of substance
If multiple amplification cycles are performed to increase DNA copy number, then the quantity of repeating segments increases, but the complexity of maintaining ORF continuity and controlling the process increases
Solution Approach 1:
The amplification process is structured as periodic cycles of digestion with SapI endonuclease followed by controlled ligation. This periodic repetition of standardized steps (digestion→ligation→transformation→selection) enables systematic increase in copy number while maintaining process control and ORF integrity through consistent application of the same protocol.
Solution Approach 2:
The system allows control of amplification parameters including the number of SapI sites introduced, the concentration of DNA fragments, and the ligation conditions. By adjusting these parameters, one can control the degree of amplification and the number of repeating units while maintaining ORF continuity through optimized reaction conditions.
3Quantity of substance
If conventional vectors are used for DNA amplification, then basic cloning capability is provided, but the ability to efficiently express and produce polyepitopic proteins deteriorates
Solution Approach 1:
The vector system integrates multiple functions into a single platform: the amplifying module enables controlled DNA repetition, the expression module (with promoter, ribosome binding site, terminator) enables protein expression, and selection markers enable clone identification. This multi-functional design simplifies the manufacturing process by combining amplification and expression capabilities in one vector system.
Solution Approach 2:
The vector is pre-designed with all necessary elements for successful amplification and expression before the actual experiment. The SapI sites are pre-positioned, the expression cassette is pre-assembled, and selection markers are pre-included. This preliminary preparation eliminates the need for complex step-by-step construction during the amplification process, making the system easier to use.
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 enables the efficient production of polyepitopic proteins with hundreds of copies, maintaining ORF continuity and facilitating the creation of improved vaccines and other applications by amplifying DNA segments to achieve the desired number of repeating units.
Implementation Method 1
two convergent DNA sequences recognised by the SapI endonuclease
Implementation Method 2
modified such that it is equipped with single-stranded sticky ends, which ensure the directional ligation
Implementation Method 3
the isolated fragment is autoligated
Implementation Method 4
directionally amplifying a cloned DNA fragment
Data Source
AI summary
The present invention is directed to a method of manufacturing a polyepitopic protein comprising the steps of cloning a blunt-ended DNA sequence by encoding the epitope that is to be cloned into a DNA vector recognized by the endonuclease SmaI or the endonuclease SapI and isolating the polyepitopic protein by transforming a bacterial host cell with such vector.