DNA Expression Vector With His-Tag and GFP for Protein Purification
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Solution Overview
Problem
Current recombinant DNA technologies face challenges in efficiently expressing and purifying enzymes and proteins for industrial and medical applications, particularly in achieving high activity under varying conditions and ensuring precise localization and purification of target proteins.
Innovation Solution
A DNA expression vector system is developed, incorporating a promoter, tag sequence, cleavage sites, and a marker gene, which includes specific sequences for enhanced expression and localization of proteins, such as the CMV promoter, His-Tag, and GFP, allowing for targeted delivery and purification of proteins using protease recognition and fluorescence tagging.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional cloning vectors are used for protein expression, then basic expression can be achieved, but expression efficiency and protein activity under varying conditions are insufficient
Solution Approach 1:
The patent employs a composite vector system combining multiple functional elements: CMV promoter for high-level transcription, His-Tag for purification, GFP for visualization, and protease cleavage sites for precise protein release. This composite structure resolves the contradiction by integrating multiple functions that collectively enhance both expression efficiency and protein activity stability under varying industrial conditions.
Solution Approach 2:
The expression vector is designed with multi-functionality to address multiple requirements simultaneously: the CMV promoter drives high-level expression in mammalian cells, the His-Tag enables purification through metal affinity chromatography, GFP provides real-time expression monitoring, and protease sites allow controlled protein release. This universal design resolves the contradiction by making a single vector system capable of achieving both high productivity and reliable protein activity across different applications.
2Manufacturing precision
If proteins are expressed without localization tags, then expression simplicity is maintained, but precise localization and purification of target proteins cannot be achieved
Solution Approach 1:
The His-Tag and GFP sequences serve as intermediary elements that mediate between the expressed protein and the purification/detection systems. The His-Tag acts as a mediator for affinity purification through nickel or cobalt columns, while GFP mediates visualization and selection. These intermediaries resolve the contradiction by enabling precise localization and purification without requiring complex structural modifications to the target protein itself.
Solution Approach 2:
The vector design segments the protein expression system into distinct functional modules: the target protein coding sequence, the His-Tag sequence for purification, the GFP sequence for visualization, and protease cleavage sites for controlled release. This segmentation allows each element to perform its specific function independently, achieving precise localization and purification while keeping the overall system manageable through modular construction.
3Adaptability or versatility
If enzymes are isolated from traditional sources, then natural enzyme activity is obtained, but adaptability to modern food production conditions is limited
Solution Approach 1:
The patent utilizes recombinant DNA technology to change the genetic parameters of enzyme production, allowing expression of enzymes with modified amino acid sequences that are optimized for industrial conditions. By altering the DNA sequence encoding the enzyme, proteins can be engineered to withstand varying temperatures, pH levels, and other industrial processing conditions, thereby resolving the contradiction between adaptability and activity consistency through rational protein design and directed evolution approaches.
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 vector system enables high-yield expression of biologically active enzymes and proteins with improved stability and localization, facilitating their use in industrial processes and medical applications, such as food processing and gene therapy, by ensuring precise targeting and efficient purification.
Implementation Method 1
a promoter configured to drive the expression of the transgene in the cell
Implementation Method 2
a tag sequence encoding a tag peptide directing the protein of the expressed transgene to a pre-determined location
Implementation Method 3
GFP (green fluorescent protein) sequence is often used as biomarker to follow the expression process. In cells where the tagged transgene is expressed, the GFP is also produced, and those cells can be observed under fluorescence microscopy
Implementation Method 4
a first cleavage sequence encoding a peptide that is recognizable by a protease
Data Source
AI summary
An expression vector is provided. The vector includes a promoter configured to drive the expression of the transgene in the cell. The vector also includes a tag sequence encoding a tag peptide directing the protein of the expressed transgene to a pre-determined location. The vector further includes a cleavage sequence encoding a peptide that is recognizable by a protease and a marker gene configured to encoding a protein to indicate the expression of the transgene.