Biopolym. Cell. 2017; 33(3):214-220.
Molecular and Cell Biotechnologies
Production, purification of the recombinant analog of Y-box-binding protein and its interaction with poly(ADP-ribose), RNA, single- and double-stranded DNAs
1Alemasova E. E., 1, 2Naumenko K. N., 1Pestryakov P. E., 1, 2Lavrik O. I.
  1. Novosibirsk Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences
    8, Akademika Lavrentieva Ave., Novosibirsk, Russian Federation, 630090
  2. Novosibirsk State University
    2, Pirogova Str., Novosibirsk, Russian Federation, 630090


Aim. Production and purification of the recombinant histidine-tagged Y-box- binding protein and study of its interaction with DNA and poly(ADP-ribose). Methods. Ligation-independent 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 protein 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.
Keywords: YB-1, protein purification, poly(ADP-ribose) (PAR), DNA repair


[1] Lyabin DN, Eliseeva IA, Ovchinnikov LP. YB-1 protein: functions and regulation. Wiley Interdiscip Rev RNA. 2014; 5(1): 95–110.
[2] D'Amours D, Desnoyers S, D'Silva I, Poirier GG. Poly(ADP-ribosylation reactions in the regulation of nuclear functions. Biochem J. 1999; 342(Pt 2): 249–68.
[3] Muthurajan UM, Hepler MR, Hieb AR, Clark NJ, Kramer M, Yao T, Luger K. Automodification switches PARP-1 function from chromatin architectural protein to histone chaperone. Proc Natl Acad Sci U S A. 2014; 111(35):12752–57.
[4] Rulten SL, Rotheray A, Green RL, Grundy GJ, Moore DA, Gómez-Herreros F, Hafezparast M, Caldecott KW. PARP-1 dependent recruitment of the amyotrophic lateral sclerosis-associated protein FUS/TLS to sites of oxida-tive DNA damage. Nucleic Acids Res. 2014; 42(1): 307–14.
[5] Teloni F, Altmeyer M. Readers of poly(ADP-ribose): designed to be fit for purpose. Nucleic Acids Res. 2016; 44(3): 993–1006.
[6] Kretov DA, Curmi PA, Hamon L, Abrakhi S, Desforges B, Ovchinnikov LP, Pastré D. mRNA and DNA selection via protein multimerization: YB-1 as a case study. Nucleic Acids Res. 2015; 43(19):9457–73.
[7] Studier FW. Stable expression clones and auto-induction for protein production in E. coli. Methods Mol Biol. 2014; 1091:17–32.
[8] Alemasova EE, Moor NA, Naumenko KN, Kutuzov MM, Sukhanova MV, Pestryakov PE, Lavrik OI. Y-box-binding protein 1 as a non-canonical factor of base excision repair. Biochim Biophys Acta. 2016; 1864(12):1631–40.