Rapid synthesis of DNA-cysteine conjugates for expressed protein ligation.
We report a rapid method for the covalent modification of commercially available amino-modified DNA oligonucleotides with a cysteine moiety. The resulting DNA-cysteine conjugates are versatile reagents for the efficient preparation of covalent DNA-protein conjugates by means of expressed protein ligation (EPL). The EPL method allows for the site-specific coupling of cysteine-modified DNA oligomers with recombinant intein-fusion proteins, the latter of which contain a C-terminal thioester enabling the mild and highly specific reaction with N-terminal cysteine compounds. We prepared a cysteine-modifier reagent in a single-step reaction which allows for the rapid and near quantitative synthesis of cysteine-DNA conjugates. The latter were ligated with the green fluorescent protein mutant EYFP, recombinantly expressed as an intein-fusion protein, allowing for the mild and selective formation of EYFP-DNA conjugates in high yields of about 60%. We anticipate many applications of our approach, ranging from protein microarrays to the arising field of nanobiotechnology.
Lovrinovic
Marina
M
Universität Dortmund, Fachbereich Chemie, Biologisch-Chemische Mikrostrukturtechnik, Otto-Hahn Str. 6, D-44227 Dortmund, Germany.
Niemeyer
Christof M
CM
D001483:Base Sequence; D002853:Chromatography, Liquid; D003545:Cysteine / Q000138:chemical synthesis* / Q000737:chemistry; D004247:DNA / Q000138:chemical synthesis* / Q000737:chemistry; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D011506:Proteins / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D017931:DNA Primers; D011506:Proteins; D004247:DNA; D003545:Cysteine