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Construction of Semisynthetic DNA-Protein Conjugates with Phi X174 Gene A* Protein.
MedLine Citation:
PMID:  22616938     Owner:  NLM     Status:  Publisher    
Abstract/OtherAbstract:
DNA-protein conjugates have frequently been used as versatile molecular tools for a variety of applications in biotechnology to harness synergistic effects of DNA and protein functions. With applications for DNA-protein conjugates growing, easy-to-use and economical methods for the synthesis of DNA-protein conjugates are required. In this study, we developed a method for site-specific labeling of single-stranded DNA (ssDNA) to a recombinant protein of interest (POI) through the Gene-A* protein (Gene-A*) from bacteriophage phi X174, without any chemical modifications of ssDNA. Gene-A* protein is an enzyme that site-selectively cleaves an oligodeoxyribonucleotide (ODN) containing a Gene-A* recognition sequence, at which point a tyrosine residue of Gene-A* is bonded to the 5'-phosphoryl group of the cleavage site via a stable phosphotyrosine linkage. Here, we constructed three kinds of recombinant proteins fused to Gene-A*: N-terminally Gene-A*-fused enhanced green fluorescent protein (EGFP), C-terminally Gene-A*-fused EGFP and N-terminally Gene-A*-fused firefly luciferase (FLuc). The reaction yields of DNA-protein conjugation catalyzed by the Gene-A* moiety reached 80-90% in the three proteins, and kinetic study revealed that the reaction achieved a steady state after ten min. Moreover, dot blot analyses were performed to evaluate the hybridization and aptamer-forming ability of ssDNA conjugated to the Gene-A* moiety of a recombinant Gene-A*-FLuc protein. This study demonstrated that a strategy using recombinant proteins fused to Gene-A* could offer a versatile, rapid, easy-to-use and economical platform for producing DNA-protein conjugates.
Authors:
Yasumasa Mashimo; Hitomi Maeda; Masayasu Mie; Eiry Kobatake
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Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2012-5-23
Journal Detail:
Title:  Bioconjugate chemistry     Volume:  -     ISSN:  1520-4812     ISO Abbreviation:  -     Publication Date:  2012 May 
Date Detail:
Created Date:  2012-5-23     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  9010319     Medline TA:  Bioconjug Chem     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
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