Efficient introduction of aryl bromide functionality into proteins in vivo

Area of Science:

• Biochemistry
• Materials Science
• Synthetic Biology

Background:

• Protein engineering offers alternatives to conventional polymers, but is limited by the 20 standard amino acids.
• Incorporating novel functional groups into proteins is challenging via traditional biosynthetic routes.

Purpose of the Study:

• To biosynthetically incorporate para-bromophenylalanine (p-Br-phe) into a model protein, mouse dihydrofolate reductase (DHFR).
• To demonstrate control over the degree of amino acid substitution.
• To explore new applications for engineered proteins.

Main Methods:

• Utilized a mutant bacterial phenylalanyl-tRNA synthetase (PheRS) with relaxed substrate specificity.
• Coexpressed the mutant PheRS and DHFR in a phenylalanine auxotrophic Escherichia coli host.
• Supplemented minimal medium with p-Br-phe to achieve amino acid replacement.

Main Results:

• Achieved 88% replacement of phenylalanine residues with p-Br-phe in DHFR.
• Demonstrated control over substitution levels by varying p-Br-phe concentration.
• Obtained protein expression yields of 20-25 mg/l.
• Confirmed stability of the aryl bromide group during purification.

Conclusions:

• Biosynthetic incorporation of p-Br-phe is feasible and controllable.
• Brominated proteins enable post-translational modification via metal-catalyzed coupling reactions.
• This approach expands the utility of engineered proteins for advanced materials and structural biology (e.g., MAD X-ray studies).