Tag-Cleavable Fusion Protein Expression Vectors for Diverse Host Systems

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Solution Overview

Problem

Current methods lack a unified expression system capable of producing a wide variety of target polypeptides efficiently, as different polypeptides require specific expression systems, such as mammalian or non-mammalian systems, and may be inactive due to terminal amino acid residues added during protein expression.

Innovation Solution

A kit containing multiple expression vectors for various systems, including E. coli, yeast, baculovirus, mammalian cells, and cell-free systems, which allow for high-efficient cloning and production of tag-cleavable fusion proteins using protein tags and protease recognition sites, ensuring precise cleavage and removal of tags to obtain the exact amino acid sequence of the target polypeptide.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Adaptability or versatility

If a single expression system is used, then the process is simple, but it cannot produce a wide variety of target polypeptides requiring different expression systems

Engineering Contradiction:

Improveability to produce different target polypeptidesVSAvoidnumber of expression vectors and systems

Core Design Contradiction:

Adaptability or versatilityVSDevice complexity

Solution Approach 1:

The patent creates a universal expression system where vectors can function across multiple host species (E. coli, yeast, mammalian cells). The vectors contain universal elements like protease recognition sites and cloning sites that work across different expression systems, allowing a single kit to produce diverse polypeptides in various hosts without requiring system-specific optimization for each target protein

Inventive Principle:

Principle #6Universality (Multi-functionality)

2Productivity

If protein tags are added to improve expression and purification, then expression efficiency increases, but the target polypeptide becomes inactive due to terminal amino acid residues

Engineering Contradiction:

Improveprotein expression efficiencyVSAvoidprotein activity

Core Design Contradiction:

ProductivityVSReliability

Solution Approach 1:

The fusion protein is segmented into distinct functional modules: N-terminal protein tag for purification, protease recognition site for controlled cleavage, and C-terminal target polypeptide. This segmentation allows the tag to serve its purification function while enabling complete removal before the target protein, preventing any terminal residues from interfering with protein activity

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

A protease recognition site acts as an intermediary element between the protein tag and target polypeptide. This intermediary sequence enables specific protease cleavage at a defined location, ensuring complete removal of the tag and prevention of unwanted terminal amino acid residues that would otherwise compromise target protein activity

Inventive Principle:

Principle #24Intermediary (Mediator)

3Manufacturing precision

If different expression systems are used for different target polypeptides, then each polypeptide can be produced correctly, but the overall process becomes complex and time-consuming

Engineering Contradiction:

Improveaccuracy of amino acid sequenceVSAvoidtime to select and switch expression systems

Core Design Contradiction:

Manufacturing precisionVSLoss of time

Solution Approach 1:

The expression vectors are pre-designed with universal elements including standardized cloning sites (EcoR I and Xho I), protease recognition sites (thrombin, FXa, TEV), and selection markers that function across multiple host species. This preliminary preparation allows researchers to clone any target gene into the appropriate vector and express it in the desired host without time-consuming system selection and optimization

Inventive Principle:

Principle #10Preliminary action

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 efficient production and purification of target polypeptides across different expression systems, minimizing inactivity issues and maximizing the diversity of polypeptides that can be produced, while ensuring the exact amino acid sequence is maintained post-cleavage.

Implementation Method 1

The protein tag can be removed by protease cleavage. Upon protease cleavage at the protease recognition site, the target polypeptide separates from the protein tag.

Methodology Applied

Scientific EffectProtease cleavage: Enzyme

Data Source

PatentUS9206431B2Kit for producing recombinant tag-cleavable fusion proteins via at least two different expression vectors allow protein production in two different species host cells

Publication Date: 2015.12.08 ACAD SINICA

AI summary

This invention features a kit containing multiple expression vectors for producing tag-cleavable fusion proteins in various expression systems, or for producing fusion proteins in E. coli inclusion bodies.