NSs Peptide Tag for Recombinant Protein Detection
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Solution Overview
Problem
Current techniques lack a protein tag system with high sensitivity and stability, especially for eukaryotic expression systems, which hinders the efficient tagging and monitoring of recombinant proteins.
Innovation Solution
A eukaryotic expression system utilizing a fusion protein with an Asia tospoviral common epitope, specifically the nss-tag, is developed, allowing for efficient tagging and monitoring of recombinant proteins by interacting with a target protein and using a monoclonal antibody for detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional protein tags (e.g., His-Tag) are used for tagging recombinant proteins, then the tagging process is simple, but the detection sensitivity and stability are insufficient
Solution Approach 1:
The patent changes the fundamental parameter of the tag system by using a peptide derived from the NSs protein of tospovirus (specifically residues 98-120, sequence: VRKPNGKNTGCKFTMHNQIFNPN) instead of conventional tags like His-Tag. This peptide tag incorporates a monoclonal antibody binding site, fundamentally altering how proteins are detected and tagged, thereby achieving superior detection sensitivity while maintaining relative simplicity in the tagging process.
Solution Approach 2:
The patent introduces a monoclonal antibody as an intermediary element that binds specifically to the NSs peptide tag. This antibody-tag interaction serves as the mediator for detecting and monitoring recombinant proteins, enabling high-sensitivity detection without requiring complex tagging machinery or multiple sequential steps.
2Measurement precision
If a protein tag system is developed for eukaryotic expression systems, then recombinant protein detection improves, but protein function may be affected
Solution Approach 1:
The patent applies local quality by placing the NSs peptide tag at a specific location (N-terminus or C-terminus) of the recombinant protein, rather than distributing tagging elements throughout the protein structure. This localized tagging approach minimizes interference with the protein's functional domains while ensuring the tag remains accessible for antibody binding, thus maintaining protein function stability.
Solution Approach 2:
The patent uses a peptide sequence (NSs residues 98-120) that is naturally derived from the tospovirus NSs protein, effectively copying a functional viral peptide sequence for use as a tag. This copied sequence retains the ability to bind monoclonal antibodies while being sufficiently short and simple not to disrupt eukaryotic protein folding or function.
3Difficulty of detecting and measuring
If the NSs peptide tag is used for monitoring protein interactions, then interaction detection becomes possible, but the system complexity increases
Solution Approach 1:
The patent makes the NSs peptide tag universal by designing it to serve multiple functions: (1) as a detection tag for recombinant proteins, (2) as a basis for creating monoclonal antibodies, and (3) as a tool for monitoring protein-protein interactions. This multi-functionality reduces the need for separate tagging systems for different applications, thereby managing system complexity while enhancing detection capability.
Data Source
AI summary
Provided are a eukaryotic expression system and its applications. The eukaryotic expression system has a recombinant plant cell. The recombinant plant cell includes a first vector and a second vector. The first vector expresses a fusion protein containing an Asia tospoviral common epitope. The fusion protein containing Asia tospoviral common epitope consists of an amino acid sequence as set forth in SEQ ID NO. 1, and a predetermined protein fragment connecting to the Asia tospoviral common epitope. The above eukaryotic expression system is useful for monitoring the interaction between proteins via use of a specific peptide to tag the predetermined protein and demonstrates high sensitivity and stability.