Recombinant Vector with Cellulose-Binding Domain for Protein Purification
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Solution Overview
Problem
Current methods for producing and isolating recombinant proteins in plants are inefficient and costly, lacking a reliable method for rapid and high-purity isolation of target proteins from plant bodies.
Innovation Solution
A recombinant vector carrying a cellulose-binding module 3 (CBM3) is used, which includes a linker peptide, an enterokinase-cleavage site, and a target protein-encoding gene, allowing for the isolation and purification of target proteins by adsorption to cellulose and subsequent cleavage with enterokinase, utilizing a protein extraction buffer solution and affinity chromatography.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional methods are used to produce and isolate recombinant proteins in plants, then protein production can be achieved, but the isolation process is inefficient and costly
Solution Approach 1:
The invention introduces a cellulose-binding domain as an intermediary element fused to the target protein. This domain acts as a mediator that enables specific binding to cellulose, facilitating easy isolation. The cellulose-binding domain serves as a bridge between the target protein and the cellulose matrix, allowing for simple and cost-effective purification through affinity binding without requiring complex isolation procedures
Solution Approach 2:
The invention extracts or removes the need for complex isolation procedures by using a simple cellulose-based affinity system. The target protein with attached cellulose-binding domain is directly extracted from plant cells through binding to cellulose, eliminating the need for multiple chromatography steps and complex purification protocols that are typically required in conventional methods
2Quantity of substance
If recombinant protein is produced in plant bodies, then large-scale production is possible, but rapid and high-purity isolation is difficult to achieve
Solution Approach 1:
The cellulose-binding domain serves as a specific intermediary that provides high-affinity binding to cellulose, enabling selective capture of the target protein from complex plant extracts. This specific interaction ensures that even at large scales, the target protein can be rapidly isolated with high purity through simple binding and washing steps, separating it from other plant proteins and contaminants
Solution Approach 2:
The invention utilizes cellulose as a porous material with high surface area and specific binding properties. The cellulose matrix provides a large capacity for binding target proteins while allowing easy flow through and washing, enabling both large-scale processing and high-purity isolation simultaneously through its physical structure and chemical properties
3Ease of manufacture
If simple isolation methods are used, then cost is reduced, but purification efficiency and purity are compromised
Solution Approach 1:
The cellulose-binding domain acts as a built-in intermediary that provides specific recognition and binding to cellulose. This specific interaction enables a simple one-step affinity purification method that achieves both operational simplicity and high purity simultaneously, as the domain ensures selective binding of the target protein while non-specific proteins are washed away during the simple washing step
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 method enables rapid, cheap, and effective separation of high-purity target proteins from plant bodies, facilitating large-scale production and industrial applications.
Implementation Method 1
a target protein which has a cellulose-binding domain bound thereto
Implementation Method 2
an enterokinase-cleavage site, and a target protein-encoding gene may be sequentially connected
Data Source
AI summary
The present invention relates to a recombinant vector carrying cellulose-binding domain 3 and a method for separating a target protein using the recombinant vector. The protein isolating method of the present invention can easily separate a fusion protein containing a target protein by using the recombinant vector to bind a transformed plant body to cellulose and can effectively isolate the target protein from a cellulose-binding domain by treating the fusion protein with enterokinase, thus being expected to find industrial applications in various fields.