Accessing isotopically labeled proteins containing genetically encoded phosphoserine for NMR with optimized...

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Phosphoserine (pSer) sites are often in disordered protein regions, complicating structural and functional analysis.
• Site-specific production of phosphorylated proteins is crucial for understanding phosphorylation-mediated regulation.
• Existing genetic code expansion methods for pSer lack protocols for isotope-enriched labeling media.

Purpose of the Study:

• To develop and optimize a method for producing site-specifically phosphorylated proteins in isotope-enriched media for NMR studies.
• To overcome challenges associated with serine auxotrophy in expression hosts.
• To enable routine access to pSer-enriched proteins for structural and functional investigations.

Main Methods:

• Adapted genetic code expansion in Escherichia coli with a ΔserB mutation for pSer incorporation.
• Optimized media composition and culture conditions to restore growth in minimal labeling media.
• Developed a high-density culture protocol to achieve high yields of labeled proteins.

Main Results:

• Successfully restored growth of BL21(DE3) ΔserB strains in isotope-labeling media.
• Achieved yields of ≥10 mg/L of homogenously labeled, phosphorylated superfolder GFP.
• Produced 15N-labeled SARS-CoV-2 Nucleocapsid protein with site-specific pSer at S188, demonstrating phosphorylation effects via NMR.

Conclusions:

• A cost-effective methodology for producing site-specifically phosphorylated proteins in isotope-enriched media has been established.
• This method overcomes previous limitations for producing pSer-containing proteins for NMR analysis.
• The approach facilitates routine structural and functional studies of protein phosphorylation.