Starch Binding Protein Tag for Thermal Stabilization

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Solution Overview

Problem

Current protein purification methods using affinity column chromatography are inconvenient, laborious, and costly, with limitations in isolating recombinant proteins in certain conditions, such as EDTA-containing samples, and existing protein tags are large and inefficient, especially when used for enzyme products in aquatic feeds, which face issues like rapid enzyme degradation and loss of activity at high temperatures.

Innovation Solution

Fusing a starch binding protein (SBP) with the target protein to form an SBP-tagged recombinant protein, expressing it using an eukaryotic host, and combining it with an SBP-binding matrix for purification and stabilization, allowing for efficient thermal stability and retention of activity in aquatic environments.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If traditional affinity column chromatography is used for protein purification, then proteins can be isolated, but the process becomes inconvenient, laborious, and costly

Engineering Contradiction:

Improveprotein isolation capabilityVSAvoidpurification process convenience

Core Design Contradiction:

ReliabilityVSEase of operation

Solution Approach 1:

The invention extracts the essential purification function from complex traditional affinity column systems by using a simple starch-based matrix that binds directly to the SBP tag. This eliminates the need for expensive pre-prepared affinity columns while maintaining purification effectiveness, making the process more convenient and cost-effective

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The invention replaces expensive, reusable affinity columns with a disposable starch-based matrix that can be easily prepared from common starch sources. This single-use approach eliminates the need for expensive column maintenance and regeneration, reducing overall operational costs and complexity

Inventive Principle:

Principle #27Cheap short-living objects (Disposable)

2Reliability

If existing protein tags are used for purification, then recombinant proteins can be isolated, but the tags are relatively large as compared to the target protein

Engineering Contradiction:

Improverecombinant protein isolationVSAvoidprotein tag size

Core Design Contradiction:

ReliabilityVSWeight of moving object

Solution Approach 1:

The invention uses a localized starch-binding domain (SBP) tag that is specifically designed to be compact and functionally concentrated. This small, localized tag provides the necessary purification functionality without the bulk of larger traditional tags, maintaining a favorable ratio between tag size and target protein size

Inventive Principle:

Principle #3Local quality

3Quantity of substance

If enzyme products are used for aquatic feed, then nutritional value is provided, but enzyme quickly flows away after feed is added into water

Engineering Contradiction:

Improveenzyme availability in feedVSAvoidenzyme loss in water

Core Design Contradiction:

Quantity of substanceVSObject-generated harmful factors

Solution Approach 1:

The invention merges the enzyme with a starch-binding purification tag and immobilizes the complex on a starch matrix. This creates a unified structure where the enzyme remains attached to the feed through starch binding, preventing it from washing away while maintaining its nutritional and functional value in aquatic feed applications

Inventive Principle:

Principle #5Merging (Combining)

4Quantity of substance

If enzyme products are used for aquatic feed, then nutritional value is provided, but enzyme activity will be destructed when feed pelleting temperature is higher than 100° C.

Engineering Contradiction:

Improveenzyme content in feedVSAvoidenzyme activity stability

Core Design Contradiction:

Quantity of substanceVSReliability

Solution Approach 1:

The invention performs preliminary thermal stabilization by fusing the heat-sensitive enzyme with the thermally stable SBP tag before feed processing. This pre-protection through tag fusion shields the enzyme from thermal degradation during high-temperature pelleting, preserving enzyme activity while maintaining feed production efficiency

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

The method provides a cost-effective, high-recovery, and thermally stable protein purification system, enabling the production of clean-cut, tag-free proteins with enhanced stability and activity retention, suitable for various hosts and applications including aquatic feeds.

Implementation Method 1

combining the SBP-tagged recombinant protein with a SBP-binding matrix

Methodology Applied

Scientific EffectAffinity binding: Adsorption

Data Source

PatentUS8986955B2Method for increasing thermal stability and retaining activity of a protein

Publication Date: 2015.03.24 SIMPSON BIOTECH CO LTD

AI summary

The present invention provides a method and a system for increasing thermal stability of a target protein comprising fusing a starch binding protein (SBP) with the target protein to form a SBP-tagged recombinant protein and combining the SBP-tagged recombinant protein with a SBP-binding matrix. The present invention also provides a method for retaining an activity of a target protein in aquatic environment comprising fusing a starch binding protein (SBP) with the target protein to form a SBP-tagged recombinant protein and combining the SBP-tagged recombinant protein with a SBP-binding matrix.