Translational Fusion Partner Screening for Recombinant Protein Secretion
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Solution Overview
Problem
Current recombinant protein expression systems struggle to efficiently produce and secrete proteins that are difficult to express using conventional methods, lacking a general technique for the secretory production of all proteins.
Innovation Solution
A rapid and efficient automatic screening method for identifying translational fusion partners (TFPs) that induce the secretion of target proteins by co-transforming reporter protein-deficient host cells with linear vectors containing nucleic acid fragments and a target protein sequence, allowing in vivo recombination and identifying cells with reporter protein activity to isolate effective TFPs.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional recombinant expression systems are used, then protein production is achieved for easily expressed proteins, but secretion is poor or nonexistent for difficult-to-express proteins
Solution Approach 1:
The invention segments the problem of identifying secretion partners by creating a library of individual nucleic acid fragments (5-20 amino acids) from secreted proteins. Each fragment is tested independently in the trap system, allowing systematic identification of effective secretion-inducing sequences for different target proteins, thereby improving both secretion efficiency and adaptability across various protein types.
Solution Approach 2:
The invention introduces a reporter protein as an intermediary in the trap system. The reporter protein serves as a mediator between the nucleic acid fragments from secreted proteins and the target protein of interest. When a nucleic acid fragment successfully induces secretion, it is detected through reporter protein activity, enabling indirect identification of effective translational fusion partners without directly observing target protein secretion.
2Measurement precision
If manual screening methods are used to identify translational fusion partners, then secretion can be detected, but the process is time-consuming and labor-intensive
Solution Approach 1:
The trap system is designed to automatically detect effective translational fusion partners through self-service mechanisms. The reporter protein automatically indicates successful secretion induction through its activity (e.g., enzymatic activity or detectable signal), eliminating the need for manual inspection and analysis. This automated detection maintains high measurement precision while dramatically reducing screening time and labor requirements.
Solution Approach 2:
The invention utilizes color changes or visual signals from reporter proteins to indicate successful secretion events. The reporter protein's detectable activity (such as colorimetric or fluorescent signals) provides immediate visual feedback, allowing rapid automated detection and sorting of cells with effective translational fusion partners, thereby reducing both time and labor intensity while maintaining detection accuracy.
3Productivity
If a library of nucleic acid fragments is screened, then effective TFPs can be identified, but the screening complexity and resource requirements increase
Solution Approach 1:
The library is segmented into small, manageable nucleic acid fragments (5-20 amino acids) rather than testing entire secreted proteins. This segmentation reduces the complexity of each individual screening unit while maintaining the ability to identify effective sequences. The fragmented approach allows parallel processing of multiple fragments, increasing identification speed without proportionally increasing overall system complexity.
Solution Approach 2:
The trap system employs a universal reporter protein that can detect secretion induction from any nucleic acid fragment in the library. This universal detection mechanism handles diverse fragments with a single standardized system, avoiding the need for multiple specialized screening systems. The multi-functional nature of the reporter system maintains high identification speed while controlling complexity through standardization.
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
Enables the secretion of target proteins, including those previously not expressed or expressed at low levels, optimizing recombinant expression and facilitating the production of proteins that are hard to produce using traditional systems.
Implementation Method 1
incubating said plurality of transformed host cells under conditions effective to allow in vivo recombination of said linear vectors and said nucleotide sequence encoding a target protein
Data Source
AI summary
The invention relates to techniques for the rapid screening of suitable translational fusion partners (TFPs) capable of inducing secretory production of recombinant proteins, especially proteins that are difficult to produce using conventional recombinant production methods.