High-throughput purification of recombinant proteins using self-cleaving intein tags

Area of Science:

• Biotechnology
• Molecular Biology
• Protein Expression

Background:

• High throughput recombinant protein production often relies on affinity tags for purification.
• Tag removal can be unpredictable and challenging, complicating downstream applications.

Purpose of the Study:

• To develop and validate high throughput methods for producing untagged recombinant proteins.
• To utilize self-cleaving intein tags for simplified and efficient protein purification.

Main Methods:

• Employed self-cleaving chitin-binding domain (CBD) and elastin-like polypeptide (ELP) intein tags.
• Purified model proteins (E. coli beta-galactosidase and sfGFP) in a 96-well filter plate format.
• Validated the method in a high throughput expression platform using 24-well plates.

Main Results:

• Achieved >90% pure untagged protein product in a single purification step.
• Demonstrated low well-to-well and plate-to-plate variability across multiple targets and tags.
• Successfully scaled down the intein purification scheme for high throughput applications.

Conclusions:

• The developed intein-based method enables reproducible production of highly pure untagged recombinant proteins.
• This approach offers a simple, cost-effective, and convenient alternative to traditional tagged protein purification.
• The microtiter plate format is suitable for high throughput screening and production.