Production, purification of the recombinant analog of Y-box-binding protein and its interaction with poly(ADP-ribose), RNA, single- and double-stranded DNAs

E. E. Alemasova, K. N. Naumenko, P. E. Pestryakov, O. I. Lavrik
2017 Biopolymers and Cell  
Aim. Production and purification of the recombinant histidine-tagged Y-box-binding protein 1 and study of its interaction with DNA, RNA and poly(ADP-ribose). Methods. Ligationindependent cloning, PCR, Sanger sequencing, protein chromatography, polyacrylamide gel electrophoresis, and electrophoresis mobility shift assay. Results. cDNA coding for the YB-1 protein has a previously undocumented two single nucleotide polymorphisms. The expression construct for production of the his-tagged YB-1
more » ... n was designed to simplify the purification procedure and an appropriate protocol for protein purification was developed. Using electrophoresis mobility shift assay, we have shown that poly(ADP-ribose) competes with a double-and single-stranded DNA and RNA for binding to purified recombinant his-tagged YB-1. Conclusions. In the present work we developed and optimized the procedure of the recombinant YB-1 protein production and purification from bacterial cells. We found that poly(ADP-ribose) at high concentration is able to recruit YB-1 protein from the YB-1-DNA and YB-1-RNA complexes, suggesting a possible YB-1 involvement in DNA repair. K e y w o r d s: YB-1, protein purification, poly(ADP-ribose) (PAR), DNA repair.
doi:10.7124/bc.000954 fatcat:34b2gkgyurefdmarvpi7kaappq