Cloning and expression of putative helikase Lhr from E.coli K12

Katsiaryna Shamayeva^a, Vitali Bialevich^a, Eva Csefalvay^a

^aDepartment of Structure and Function of Proteins, Institute of Systems Biology and Ecology, Academy of Sciences of the Czech Republic, and Institute of Physical Biology, University of South Bohemia in Ceske Budejovice, Zamek 136, CZ-373 33 Nove Hrady, Czech Republic

Gene lhr is widely distributed in the E .coli chromosomes, and encoding for of 1,538 amino acids, making it the longest known protein in E. coli. Absence of this gene is not essential for E.coli growth under various laboratory conditions. The gene (tentatively termed lhr for long helicase related) contains the seven conserved motifs of the DNA and RNA helicase superfamily II, which seems like Lhr protein is probable very large helicase [1].

The polymerase chain reaction (PCR) technique has been used to obtain lhr gene from E. coli K12 genomic DNA. Optimal conditions for PCR have been found. Lhr gene has been cloned into 2 types of commercial plasmids (pBluescript and pET32a) to obtain expression system able to produce Lhr protein. Expression of Lhr has been tested in several E.coli expression strains.

The aim of our work is to produce and prepare Lhr protein in the amount sufficient for structural studies.

1. Nina Bacher Reuven, Eugene V. Koonin, Kenneth E. Rudd, and Murray P. Deutscher (1995), The Gene for the Longest Known Escherichia coli Protein is a Member of Helicase Superfamily II, Journal of Bacteriology, Oct. 1995, p. 5393–5400

Acknowledgements:

This work is supported by the Ministry of Education of the Czech Republic (MSM6007665808 and LC06010) and by the Academy of Sciences of the Czech Republic (AVOZ60870520).