C-terminal modifications of a protein by UAG-encoded incorporation of puromycin during in vitro protein synthesis in the absence of release factor 1.
Deactivation of release factor 1 by polyclonal antibodies in an in vitro translation system, which was used to express the esterase gene, led to the reversible elimination of naturally occurring termination. This technique allowed the antibiotic puromycin to be used as an acceptor substrate for the peptidyl residue in the peptidyl-transferase reaction. This resulted in more than 80 % yield of protein with C-terminally incorporated puromycin. pCpPuromycin that was either conjugated with the Cy3 fluorophor or biotin by N4 alkylation of cytosine, also acted as an acceptor substrate for the peptidyl-transferase reaction and was incorporated into the protein C terminus. The resulting conjugates possessed Cy3-specific fluorescence and affinity to streptavidin-coated surfaces, respectively. This left the enzymatic activity of the reporter protein unaffected. It was also shown that extension of puromycin on its 5'-hydroxyl end by up to ten deoxyoligonucleotides also allowed conjugation with the C terminus of in vitro translated protein when RF1-dependent termination was suppressed. However, the conjugation yield decreased upon addition of more than six nucleotides.
Agafonov
Dmitry E
DE
Fellow of the Alexander von Humboldt Foundation on leave from the Institute of Protein Research, Russian Academy of Sciences, Pushchino, Russia.
Rabe
Kersten S
KS
Grote
Michael
M
Voertler
C Stefan
CS
Sprinzl
Mathias
M
D000818:Animals; D001710:Biotin / Q000378:metabolism; D002474:Cell-Free System; D018388:Codon, Terminator; D004950:Esterases / Q000235:genetics / Q000378:metabolism; D015394:Molecular Structure; D010443:Peptide Chain Termination, Translational; D010454:Peptide Termination Factors / Q000235:genetics / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D011691:Puromycin / Q000737:chemistry* / Q000378:metabolism; D019809:Streptavidin / Q000378:metabolism
D018388:Codon, Terminator; D010454:Peptide Termination Factors; D010455:Peptides; D011691:Puromycin; D001710:Biotin; D019809:Streptavidin; D004950:Esterases