Preparation of recombinant proteins RhsA and RhsB from E.coli

Vitali Bialevich1, Eva Csefalvay1

 

1Department 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

 

Genes rhsA and rhsB are widely distributed in the E. coli K12 chromosome. RhsA and RhsB proteins are products of these genes and have unknown functions and structures. There is the theory that protein RhsA is required for the maximal biosynthesis of the K5 polysaccharide [1].

The main objective of our project is to get the crystals of RhsA and RhsB proteins for synchrotron diffraction experiments. This work includes next steps: to construct the vectors containing genes rhsA and rhsB and to build them in bacterial DNA, to get RhsA and RhsB proteins, and finally to prepare proteins for crystallization experiments. The polymerase chain reaction (PCR) technique has been used to obtain rhsA and rhsB templates from bacterial DNA. Optimal conditions for PCR have been found, and structures of expression systems are under solution. 

References:

[1] C. McNulty, J. Thompson, B. Barrett, L. Lord, C. Andersen and I. S. Roberts. M. Microbiology (2006) 59 (3), 907–922.

 

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).