Revealing the amino acid composition of proteins within an expanded genetic code.
The genetic code can be manipulated to reassign codons for the incorporation of non-standard amino acids (NSAA). Deletion of release factor 1 in Escherichia coli enhances translation of UAG (Stop) codons, yet may also extended protein synthesis at natural UAG terminated messenger RNAs. The fidelity of protein synthesis at reassigned UAG codons and the purity of the NSAA containing proteins produced require careful examination. Proteomics would be an ideal tool for these tasks, but conventional proteomic analyses cannot readily identify the extended proteins and accurately discover multiple amino acid (AA) insertions at a single UAG. To address these challenges, we created a new proteomic workflow that enabled the detection of UAG readthrough in native proteins in E. coli strains in which UAG was reassigned to encode phosphoserine. The method also enabled quantitation of NSAA and natural AA incorporation at UAG in a recombinant reporter protein. As a proof-of-principle, we measured the fidelity and purity of the phosphoserine orthogonal translation system (OTS) and used this information to improve its performance. Our results show a surprising diversity of natural AAs at reassigned stop codons. Our method can be used to improve OTSs and to quantify amino acid purity at reassigned codons in organisms with expanded genetic codes.
Department of Cellular & Molecular Physiology, Yale University, New Haven, CT 06520, USA Systems Biology Institute, Yale University, West Haven, CT 06516, USA.
Hans R
http://orcid.org/0000-0001-9477-2949
HR
Aerni
Keck Biotechnology Resource Laboratory, Yale University, New Haven, CT 06511, USA.
Mark A
MA
Shifman
Department of Cellular & Molecular Physiology, Yale University, New Haven, CT 06520, USA Systems Biology Institute, Yale University, West Haven, CT 06516, USA.
Svetlana
S
Rogulina
Departments of Biochemistry and Chemistry, The University of Western Ontario, London, ON N6A 5C1, Canada.
Patrick
P
O'Donoghue
Department of Cellular & Molecular Physiology, Yale University, New Haven, CT 06520, USA Systems Biology Institute, Yale University, West Haven, CT 06516, USA jesse.rinehart@yale.edu.
Jesse
J
Rinehart
D000596:Amino Acids / Q000032:analysis* / Q000378:metabolism; D018388:Codon, Terminator; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics; D017353:Gene Deletion; D010454:Peptide Termination Factors / Q000235:genetics; D010768:Phosphoserine / Q000378:metabolism; D014176:Protein Biosynthesis; D011506:Proteins / Q000737:chemistry / Q000235:genetics*; D040901:Proteomics / Q000379:methods*
D000596:Amino Acids; D018388:Codon, Terminator; D029968:Escherichia coli Proteins; D010454:Peptide Termination Factors; D011506:Proteins; C489005:prfA protein, E coli; D010768:Phosphoserine