Split HaloTag Reconstitution via Orthogonal Peptide Tags
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Solution Overview
Problem
Current methods for endogenous protein tagging, such as CRISPR/Cas9-mediated genome engineering, face challenges with large fluorescent protein tags that perturb protein function and efficiency, and split fluorescent protein systems like FP11 tags have limitations in fluorophore brightness and photostability, making it difficult to detect low expression targets and requiring additional recruitment strategies.
Innovation Solution
A tag-assisted split enzyme complementation system using orthogonal peptide tags and their binders to scaffold the complementation of split reporters like HaloTag, allowing for efficient detection of protein proximity and interactions without the need for large tags, enabling the use of bright and photostable organic fluorophores.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Illumination intensity
If large fluorescent protein tags are used for endogenous protein tagging, then fluorescence detection is enabled, but the tagged protein's localization and function are perturbed and knock-in efficiency is reduced
Solution Approach 1:
The fluorescent protein is divided into two separate peptide tags (FP1-10 and FP11) that can be independently knocked into the genome. This segmentation allows for smaller individual tags that cause less perturbation to the target protein while still enabling fluorescence detection when both tags are present and in proximity.
Solution Approach 2:
The invention introduces peptide tags as intermediary elements that mediate between the target protein and the fluorescent signal. These small peptide tags serve as recruitment sites for the split fluorescent protein fragments, enabling indirect fluorescence labeling that minimizes direct interference with the target protein's function.
2Length of moving object
If FP11 tags are used for protein detection, then small size and fluorescence are achieved, but fluorophore brightness and photostability are insufficient for detecting low expression targets
Solution Approach 1:
The invention merges the advantages of small peptide tags with bright fluorescent proteins by creating a system where two small peptide tags recruit split fluorescent protein fragments. The resulting reconstituted fluorescent protein provides high brightness and photostability while the peptide tag system maintains minimal size and perturbation.
3Adaptability or versatility
If split protein complementation systems other than FP1-10/11 are used, then diverse reporter outputs are enabled, but additional protein-recruitment strategies are required and the split points are not suitable for short peptide tagging
Solution Approach 1:
The invention creates a universal peptide tag system that can be applied to multiple split protein complementation systems (FP, luciferase, HaloTag, protease). The orthogonal peptide tags and their binders serve as a universal recruitment mechanism that simplifies the adaptation of diverse reporter systems to endogenous protein tagging.
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 system enhances the detection of low expression targets and protein-protein interactions with improved signal-to-background ratios, allowing for the sorting of biallelic cells and multiplexing capabilities, outperforming traditional split GFP systems in terms of brightness and background reduction.
Implementation Method 1
a first detection fusion protein comprising a first portion of a split reporter and a first affinity agent that specifically binds to the first peptide tag
Implementation Method 2
the first portion of the split reporter and the second portion of the split reporter produce a first signal when in proximity
Data Source
AI summary
We have developed the tag-assisted split enzyme complementation (TASEC) approach, which uses two orthogonal small peptide tags and their cognate binders to conditionally drive complementation of a split enzyme upon labeled protein expression. Using this approach, we have engineered and optimized the tag-assisted split HaloTag complementation system (TA-splitHalo) and demonstrated its versatile applications in improving the efficiency of knock-in cell enrichment, detection of protein-protein interaction, and isolation of biallelic gene edited cells through multiplexing.