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A Universal Tag To Supercharge Proteins for Cytosolic Delivery
Yanan Quan1, Weina Jing1, Lei Peng1
1Department of Pharmaceutical Engineering, School of Pharmaceutical Sciences, Wuhan University, Wuhan 430071, Hubei, China.
Researchers developed a novel tagging strategy to create supercharged proteins for enhanced cellular delivery. This method, using a CBB-bearing peptide, efficiently delivers functional proteins to the cytosol without mutagenesis, outperforming existing techniques.
Area of Science:
- Biotechnology
- Protein Engineering
- Cellular Delivery
Background:
- Supercharged proteins possess high positive charges enabling efficient cellular entry.
- Current methods for creating supercharged proteins involve extensive mutagenesis, which is time-consuming and protein-limited.
Purpose of the Study:
- To develop a mutagenesis-free strategy for supercharging proteins.
- To enhance cytosolic delivery of functional proteins using a novel tagging system.
Main Methods:
- A cationic peptide (CBB-R8) bearing Coomassie brilliant blue (CBB) was synthesized.
- CBB-R8 was used to tag proteins, creating supercharged variants without altering the protein's genetic sequence.
- The charge density and delivery efficiency of tagged proteins were optimized by adjusting the tag/protein molar ratio.
Main Results:
- The CBB-R8 tagging strategy successfully supercharged proteins, enabling potent cytosolic delivery.
- Tagged proteins demonstrated superior delivery efficiency compared to a benchmark supercharged protein (+36GFP).
- Delivered proteins accessed the cytosol directly, bypassing most endocytic pathways, and retained their biological functions post-delivery.
Conclusions:
- The plug-and-charge methodology provides a simple and versatile approach to generate supercharged proteins.
- This strategy significantly facilitates protein preparation and broadens applications for intracellular delivery and manipulation.
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