Cloning and expression of rhsA and rhsB genes from E.coli

Vitali Bialevich, Eva Csefalvay

 

Department 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

 

RhsA and RhsB proteins are products of genes rhsA and rhsB accordingly. These proteins 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 RhsA and RhsB proteins in purity sufficient for biophysical 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 biophysical experiments. The polymerase chain reaction (PCR) technique has been used to obtain DNA fragments encoding for rhsA and rhsB genes from genomic DNA of E. coli K12. Optimal conditions for PCR have been found. Both rhsA and rhsB have been cloned into pBluescript SK+ and pET32a(+): the clones contained good expression system have been selected by restriction analysis and sequencing. Several expression E. coli strains have been used to test expression under several cultivation conditions.

References:

[1] C. McNulty, J. Thompson, B. Barrett, L. Lord, C. Andersen and I. S. Roberts. M. Microbiology (2006) 59 (3), 907922.

 

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).