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Utilizing a Baculovirus/Insect Cell Expression System and Expressed Protein Ligation (EPL) for Protein Semisynthesis
1Department of Cancer Biology and Genetics, the Comprehensive Cancer Center, College of Medicine, The Ohio State University, Columbus, Ohio.
Current Protocols
|January 19, 2022
Summary
Protein semisynthesis enables creating modified proteins by linking synthetic peptides and recombinant fragments. This study details a baculovirus/insect cell method for producing modified human enzymes, like phosphorylated Akt1, for functional studies.
Area of Science:
- Biochemistry
- Molecular Biology
- Chemical Biology
Background:
- Protein semisynthesis facilitates native protein reconstruction via chemoselective ligation of peptides and protein fragments.
- Incorporating site-specific post-translational modifications (PTMs) via semisynthesis enhances understanding of protein function and regulation.
- Bacterial expression systems often struggle with high yields and functional integrity of recombinant human enzymes, limiting semisynthesis applications.
Purpose of the Study:
- To present a robust protocol for protein semisynthesis using a baculovirus/insect cell expression system.
- To enable the production of proteins with site-specific chemical modifications, overcoming limitations of bacterial expression.
- To demonstrate the application of this method for generating and analyzing modified human enzymes, exemplified by phosphorylated Akt1.
Main Methods:
- Utilized a baculovirus/insect cell expression system for producing protein fragments.
- Employed expressed protein ligation (EPL) for chemoselective ligation of protein fragments and synthetic peptides.
- Developed protocols for generating C-terminal protein thioesters and purifying ligation products.
Main Results:
- Successfully produced workable levels of proteins of interest with site-specific modifications.
- Generated protein kinase Akt1 with site-specific phosphorylations in its C-terminal tail.
- Established detailed protocols for protein thioester generation, EPL, and purification.
Conclusions:
- The described baculovirus/insect cell-based protein semisynthesis and EPL strategy effectively produces modified proteins.
- This approach facilitates the study of human enzymes and their post-translational regulation, particularly for challenging targets.
- The methods are expected to increase the adoption of protein semisynthesis for investigating cell signaling pathways.
