N-terminal labeling of proteins using initiator tRNA.
Methodology based on tRNA mediated protein engineering is described for the introduction of fluorophores and other labels at the N-terminus of proteins produced in cell-free translation systems. One method for low-level (trace) N-terminal labeling is based on the use of an Escherichia coli initiator tRNA(fMet) misaminoacylated with methionine modified at the alpha-amino group. In addition to the normal formyl group, the protein translational machinery incorporates the fluorophore BODIPY-FL and the affinity tag biotin at an N-terminal end of the nascent protein. A second method for higher N-terminal labeling uses a chemically aminoacylated amber initiator suppressor tRNA and a DNA template which contains a complementary amber (UAG) codon instead of the normal initiation (AUG) codon. This more versatile approach is demonstrated using a variety of N-terminal markers including fluorescein, biotin, PC-biotin, and a novel dual marker conjugate (Biotin/BODIPY-FL).
Olejnik
Jerzy
J
AmberGen, 1106 Commonwealth Avenue, Boston, MA 02215, USA.
Gite
Sadanand
S
Mamaev
Sergey
S
Rothschild
Kenneth J
KJ
D004926:Escherichia coli / Q000235:genetics; D005456:Fluorescent Dyes / Q000737:chemistry; D014176:Protein Biosynthesis; D015202:Protein Engineering / Q000379:methods*; D011506:Proteins / Q000737:chemistry*; D012343:RNA, Transfer / Q000737:chemistry; D012358:RNA, Transfer, Met / Q000737:chemistry*; D046249:Transfer RNA Aminoacylation
D005456:Fluorescent Dyes; D011506:Proteins; D012358:RNA, Transfer, Met; D012343:RNA, Transfer