Bacteriophytochrome Peptide Tag for Specific Protein Detection
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Solution Overview
Problem
Conventional epitope tagging systems face challenges with non-specific reactions due to the amino acid sequences of tags like c-myc and FLAG, which can interfere with the isolation and confirmation of specific target proteins, and existing peptide tags can influence the three-dimensional structure and biological activity of target proteins.
Innovation Solution
Development of novel peptide tags derived from bacteriophytochrome (BphP) and a monoclonal antibody specifically recognizing these tags, allowing for effective detection and purification of target proteins without altering their structure or function.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional epitope tags (c-myc, FLAG) are used for protein detection and purification, then the target protein can be detected and purified, but non-specific reactions occur due to the amino acid sequences of the tags interfering with the isolation and confirmation of specific target proteins
Solution Approach 1:
The patent extracts the problematic amino acid sequences (c-myc, FLAG) that cause non-specific reactions and replaces them with a novel peptide tag sequence (SEQ ID NO: 1) derived from bacteriophytochrome. This extraction of harmful elements eliminates non-specific reactions while maintaining the ability to detect and purify target proteins specifically.
Solution Approach 2:
The patent changes the amino acid sequence parameter of the epitope tag from conventional sequences (c-myc, FLAG) to a novel sequence (SEQ ID NO: 1) with different biochemical properties. This parameter change results in a tag that does not cause non-specific reactions while still providing specific binding sites for antibodies.
2Reliability
If existing peptide tags are used for protein detection, then target proteins can be detected, but the tags may influence the three-dimensional structure and biological activity of target proteins
Solution Approach 1:
The patent applies local quality by designing a peptide tag (SEQ ID NO: 1) with specific local amino acid properties that minimize interaction with the target protein's three-dimensional structure. The tag is positioned at the N-terminus and has biochemical characteristics that reduce structural interference while maintaining detection specificity.
Solution Approach 2:
The patent uses a short peptide tag sequence (9 amino acids) that can be easily attached and removed without long-term structural consequences. The tag serves its detection purpose and can be cleaved from the target protein without affecting the protein's biological activity, similar to using disposable tags that complete their function and are then discarded.
Data Source
AI summary
There are provided peptide tags derived from bacteriophytochrome (BphP) that is photoreceptor protein of Deinococcus radiodurans, an antibody capable of specifically recognizing the peptide tags, hybridoma cell lines capable of producing the antibody, and uses thereof. The novel peptide tag has advantages in that it has a short length and can remove a non-specific reaction of the conventional c-myc tag and FLAG tag. Therefore, in the case of using the novel peptide tag and antibody thereto, the fusion protein expressed in a recombinant cell can be very effectively detected or purified. In addition, an epitope tagging system including the novel peptide tag and antibody thereto can be applied in various fields such as a determination of an intracellular site, a confirmation of functionality, detection and purification of specific protein, and researches on interaction between proteins.