Synthesis of covalent DNA-protein conjugates by expressed protein ligation.
Semisynthetic DNA-protein conjugates are versatile tools for many applications in bioanalytics and nanobiotechnology. We here report a method based on expressed protein ligation (EPL) for the site-specific coupling of cysteine-modified DNA oligomers with recombinant intein-fusion proteins. The latter contain a C-terminal thioester, enabling the mild and highly specific reaction with N-terminal cysteine compounds. To conveniently couple commercially available DNA oligomers with cysteine groups a universal chemical modifier was developed, containing a protected cysteine and an amino-reactive N-hydroxysuccinimide group connected by a hexaethyleneglycol moiety. Using maltose-binding protein (MBP) and green fluorescent protein mutant EYFP as a model systems, we demonstrate the feasibility of this approach, as well as the integrity and functionality of the DNA-protein conjugates synthesized. We anticipate that our concept will enable many applications, such as the generation of large arrays of surface-bound, recombinant proteins assembled by means of DNA-directed immobilization.
Lovrinovic
Marina
M
Universitat Dortmund, Fachbereich Chemie, Biologisch-Chemische Mikrostrukturtechnik, Otto-Hahn Str. 6, D-44227 Dortmund.
Spengler
Mark
M
Deutsch
Carl
C
Niemeyer
Christof M
CM
D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics; D003545:Cysteine / Q000737:chemistry*; D004247:DNA / Q000737:chemistry*; D005240:Feasibility Studies; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics; D047668:Inteins; D058619:Maltose-Binding Proteins; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D013388:Succinimides / Q000737:chemistry
D002352:Carrier Proteins; D058619:Maltose-Binding Proteins; D009841:Oligonucleotides; D011993:Recombinant Fusion Proteins; D013388:Succinimides; D049452:Green Fluorescent Proteins; D004247:DNA; D003545:Cysteine; C001426:N-hydroxysuccinimide