Synthesis of covalent DNA-protein conjugates by expressed protein ligation
| Autor: | Marina Lovrinovic, Christof M. Niemeyer, Mark Spengler, Carl Deutsch |
|---|---|
| Rok vydání: | 2006 |
| Předmět: |
Recombinant Fusion Proteins
Green Fluorescent Proteins Oligonucleotides Succinimides Thioester Maltose-Binding Proteins law.invention Green fluorescent protein Inteins chemistry.chemical_compound law Nanobiotechnology Moiety Cysteine Molecular Biology chemistry.chemical_classification Chemistry DNA Combinatorial chemistry Biochemistry Covalent bond Recombinant DNA Feasibility Studies Carrier Proteins Biotechnology |
| Zdroj: | Molecular bioSystems. 1(1) |
| ISSN: | 1742-206X |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|