Fusion Tag for Protein Solubility and Expression

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Solution Overview

Problem

Current methods for producing recombinant proteins in E. coli often result in low yields due to protein degradation and insolubility, especially for small proteins, requiring time-consuming and costly refolding processes, and lack a fusion tag that effectively enhances water solubility and expression levels.

Innovation Solution

A recombinant vector is developed with a fusion tag containing the PLX1DLGX2E domain from α-carbonic anhydrase, which is operably linked to a target protein gene, allowing for increased water solubility and expression levels without interfering with the protein's functional properties, and is particularly stable when used with the trncNEXT tag.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Stability of the object's composition

If conventional fusion tags (MBP, GST, Fh8) are used to increase water solubility, then solubility is improved, but expression level and thermal stability are insufficient

Engineering Contradiction:

Improvethermal stabilityVSAvoidexpression level

Core Design Contradiction:

Stability of the object's compositionVSProductivity

Solution Approach 1:

The patent creates a composite fusion tag by combining the NEXT tag sequence with the Fh8 tag sequence. This composite tag integrates the solubility-enhancing capability of Fh8 with the thermal stability properties of NEXT, achieving both high expression levels and excellent thermal stability that neither tag alone can provide

Inventive Principle:

Principle #40Composite materials

Solution Approach 2:

The patent modifies the fusion tag by truncating the C-terminus of the NEXT tag to create trncNEXT, optimizing the sequence composition to achieve the ideal balance between solubility and thermal stability. This parameter optimization allows the tag to maintain high expression levels while significantly improving thermal stability compared to conventional tags

Inventive Principle:

Principle #35Parameter changes

2Reliability

If refolding processes are used to convert insoluble protein aggregates into active form, then protein activity is recovered, but time and cost increase significantly

Engineering Contradiction:

Improveprotein activityVSAvoidrefolding time

Core Design Contradiction:

ReliabilityVSLoss of time

Solution Approach 1:

The fusion tag is designed to prevent insolubility in the first place by maintaining the target protein in a soluble state during overexpression in E. coli. This preliminary prevention eliminates the need for subsequent refolding processes, saving significant time and resources while ensuring protein activity

Inventive Principle:

Principle #10Preliminary action

Solution Approach 2:

The fusion tag acts as an intermediary that facilitates proper folding and solubility of the target protein during expression. By serving as a solubility-enhancing partner, it mediates the folding process to produce soluble, active protein directly, avoiding the need for denaturation and refolding steps

Inventive Principle:

Principle #24Intermediary (Mediator)

Data Source

PatentUS11542491B2Fusion tag for increasing water solubility and expression level of target protein and uses thereof

Publication Date: 2023.01.03 INDUSTRYACADEMIC COOPERATION FOUNDATION GYEONGSANG NATIONAL UNIVERSITY

AI summary

A fusion tag according to an embodiment of the present invention may increase the water solubility and expression level of a target protein. As the water solubility and expression level of a target protein in host cell can be increased by a recombinant vector including the fusion tag, the fusion tag can be advantageously used in industry.