Galectin CRD Lectin Tag for Recombinant Protein Purification
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Solution Overview
Problem
Current methods for purifying recombinant proteins are complex, have low yield, and often result in incomplete purity due to issues like co-purification of contaminants, use of toxic compounds, and modifications to the proteins, especially when using metal ion-based or other affinity chromatography techniques.
Innovation Solution
A single-step purification process utilizing the lectin domain of galectin, specifically the CRDSAT domain, as a fusion partner with a protein of interest, allowing for affinity chromatography on a lactose-grafted resin, which is specific, reversible, and does not require toxic compounds, enabling high yield and purity without modifying the protein activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If IMAC technology with histidine tag is used to purify recombinant proteins, then purification can be achieved through interaction with divalent metal ions, but the method leads to co-purification of contaminants and requires additional purification steps
Solution Approach 1:
The invention introduces a lectin domain as an intermediary purification tag that mediates specific binding to carbohydrate-containing contaminants through lectin-carbohydrate interactions. This intermediary tag enables selective capture of target proteins while leaving non-carbohydrate-containing contaminants behind, thereby improving purity without compromising productivity
Solution Approach 2:
The invention changes the binding parameter from metal ion interaction (IMAC) to lectin-carbohydrate recognition. By utilizing the specific lectin domain's ability to recognize carbohydrate structures, the method achieves higher selectivity and purity while maintaining efficient purification through affinity chromatography
2Ease of operation
If nickel ions are used in IMAC purification, then affinity chromatography can be performed, but nickel is allergenic, fetotoxic and harmful for the environment
Solution Approach 1:
The invention extracts and eliminates toxic metal ions from the purification system by replacing nickel-based IMAC with a lectin-domain-based affinity chromatography system. The lectin domain tag enables metal-free purification, removing harmful factors while maintaining ease of operation through similar affinity chromatography principles
Solution Approach 2:
The invention uses a disposable lectin domain tag that can be easily cleaved off after purification. This temporary tag enables the purification process without requiring persistent toxic materials, and can be removed by proteolytic cleavage to leave the target protein free of harmful contaminants
3Productivity
If imidazole is used to elute fusion proteins in IMAC purification, then proteins can be released from the resin, but imidazole is fetotoxic and reprotoxic
Solution Approach 1:
The invention extracts and eliminates toxic imidazole from the elution process by using lectin-carbohydrate interaction for protein release. Instead of using imidazole to compete for metal ion binding, the method uses specific lectin-carbohydrate recognition that can be reversed by changing carbohydrate concentration, thereby achieving toxic-free elution
Solution Approach 2:
The invention uses a carbohydrate molecule as a copy or analog of the natural ligand for the lectin domain. By introducing a competing carbohydrate during elution, the fusion protein is released from the resin without requiring toxic imidazole, maintaining productivity while eliminating harmful factors
4Manufacturing precision
If MBP tag is used for purification, then affinity for amylose enables purification, but steric hindrance due to molecular weight makes fusion proteins difficult to cleave
Solution Approach 1:
The invention applies local quality by using a smaller, more localized lectin domain tag compared to the large MBP tag. This localized tag provides sufficient affinity for purification while minimizing steric hindrance, enabling easier cleavage and reducing complexity without sacrificing purification specificity
Solution Approach 2:
The invention segments the purification tag into a smaller lectin domain that can be easily separated from the target protein. This segmentation allows for simpler cleavage procedures and reduces the complexity of the overall system while maintaining effective affinity purification
5Manufacturing precision
If GST tag is used for purification, then affinity for glutathione enables purification, but the affinity for glutathione Sepharose resin is low
Solution Approach 1:
The invention changes the binding parameter by using lectin-carbohydrate interaction instead of GST-glutathione interaction. The lectin domain provides strong and specific binding to carbohydrate-containing structures, increasing binding affinity and productivity while maintaining purification specificity through selective lectin-carbohydrate recognition
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 achieves high specificity and yield with a single affinity chromatography step, ensuring purity greater than 95% and allowing for easy elimination of the lectin tag, thus avoiding the limitations of existing methods, including the use of toxic substances and protein modifications.
Implementation Method 1
the lectin domain of a galectin or a part of the domain, which retains its ability to bind lactose
Implementation Method 2
purification by affinity chromatography on resins at low cost, based on the use of lectins as tag
Implementation Method 3
a sequence including a site for cleavage by a protease
Data Source
AI summary
The present invention relates to a novel method for the affinity purification of proteins of interest in a single step, based on the lectin activity of the CRD (Carbohydrate Recognition Domain) of a galectin or part of said domain retaining the ability to bind β-galactosidase derivative.