A flexible codon in genomically recoded Escherichia coli permits programmable protein phosphorylation

Area of Science:

• Biochemistry
• Molecular Biology
• Synthetic Biology

Background:

• Investigating protein phosphorylation is crucial for understanding cellular signaling.
• Current methods struggle with efficient production of specific phosphorylated and non-phosphorylated protein forms.

Purpose of the Study:

• To develop a method for site-specific production of serine- or phosphoserine-containing proteins.
• To enable the synthesis of proteins with programmable phosphorylation patterns.

Main Methods:

• Utilized a genomically recoded E. coli strain with a flexible UAG codon.
• Produced site-specific serine- or phosphoserine-containing proteins from a single DNA template.
• Synthesized human MEK1 kinase with controlled phosphorylation states.

Main Results:

• Achieved protein purities approaching 90%.
• Demonstrated programmable kinase activity in synthesized MEK1 kinase.
• Successfully produced proteins with specific serine or phosphoserine residues from one DNA template.

Conclusions:

• The developed method allows efficient, site-specific production of proteins with defined phosphorylation.
• This technique facilitates the study of the phosphoproteome by providing access to specific protein forms.
• Programmable protein phosphorylation holds potential for advancing structural and functional phosphoproteomics.