Overexpression, purification and crystallization attempts of the recombinant R subunit EcoAI


M. Lapkouski and E. Jindrova


Institute of Physical Biology, University of South Bohemia, Zamek 136, CZ-37333 Nove Hrady,

Czech Republic

Email lapkouski@greentech.cz


Type I DNA restriction enzymes are large molecular machines comprising DNA methyltransferase, ATPase, DNA translocase and endonuclease activities, essential for the antivirus defence in bacterial cells. The ATPase, DNA translocase and endonuclease activities are specified by the restriction (R) subunit of the enzyme. Our work is focused mainly on revealing structural features of the R subunit of the endonuclease EcoAI from Escherichia coli. Having optimized purification methods, crystallization trials were performed as a step for farther structural characterization of the enzyme.


1,  Jindrova, E., Schmid-Nuoffer, S., Hamburger, F., Janscak, P., Bickle, T.A, On the DNA cleavage mechanism of

     Type I restriction enzymes Nucleic Acids Res., 33, (2005),1760–1766.

2,  Janscak, P. and Bickle, T.A, The DNA recognition subunit of the type IB restriction–modification enzyme EcoAI

     tolerates circular permutations of its polypeptide chain J. Mol. Biol., 284, (1998), 937–948.

3,  Janscak, P. and Bickle, T.A, DNA supercoiling during ATP-dependent DNA translocation by the type restriction

     enzyme EcoAI J. Mol. Biol., 295, (2000), 1089–109.

4,  Murray, N.E, Type I restriction systems: sophisticated molecular machines (a legacy of Bertani and Weigle)       

     Microbiol. Mol. Biol. Rev., 64, (2000), 412–434.



This research was supported by the Ministry of Education, Youth and Sports of the Czech Republic

(MSM6007665808, LC06010) and by the Academy of Sciences of the Czech Republic (Institutional

research concept AVOZ60870520)