Microtiter plate-based screening for the optimization of DNA-protein conjugate synthesis by means of expressed protein ligation.
We report a rapid microtiter plate screening assay for the optimization of the synthesis 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 containing a C-terminal thioester that enables a mild and highly specific reaction with N-terminal cysteine compounds. To screen for optimal reaction conditions, we developed a microtiter plate-based assay that utilizes DNA-directed immobilization of the products formed in the ligation reaction of cysteine-modified DNA oligonucleotides with the model protein thioester of the maltose-binding protein (MBP), recombinantly expressed as an intein-fusion protein in E. coli. The screening assay allowed the rapid quantitative monitoring of various reaction parameters, such as the ratio of the reactants, reaction times, pH and ion strength of the buffer, the influence of various thiol additives and the nature of the chemical linker within the cysteine-bearing DNA oligonucleotide. As the consequence of the assay-based optimization, the ligation of MBP with the oligonucleotide was improved to near quantitative yields.
Lovrinovic
Marina
M
Universität Dortmund, Fachbereich Chemie, Biologisch-Chemische Mikrostrukturtechnik, Otto-Hahn Strasse 6, 44227 Dortmund, Germany.
Niemeyer
Christof M
CM
D001671:Biochemistry / Q000379:methods*; D002352:Carrier Proteins / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D004247:DNA / Q000737:chemistry*; D004926:Escherichia coli / Q000378:metabolism; D004952:Esters / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D047668:Inteins; D007477:Ions; D058619:Maltose-Binding Proteins; D008956:Models, Chemical; D009841:Oligonucleotides / Q000737:chemistry; D014176:Protein Biosynthesis; D011506:Proteins / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry
D002352:Carrier Proteins; D004952:Esters; D007477:Ions; D058619:Maltose-Binding Proteins; D009841:Oligonucleotides; D011506:Proteins; D011994:Recombinant Proteins; D004247:DNA; D003545:Cysteine