Architectural factor of bacterial nucleoid, H-NS: overproduction and verification of its functional activity

Abstract

The vector for overproduction of the H-NS protein has been constructed by means of molecular
cloning. Being one of the major structural proteins of bacterial nucleoid, it also regulates expression of some
genes. Resulting vector was transferred into E. coli BL21(DE3)∆hns strain and conditions optimal for
overproduction of the recombinant H-NS possessing six histidine residues on its C-terminal end were
estimated. Effective H-NS production was confirmed by electrophoretic fractionating of the proteins from
cell lysates. The correctness of his-tag positioning was testified by specific sorption of anti-his polyclonal
antibodies produced in rabbit. Functional activity of the protein was estimated by its ability to form specific
complexes with the DNA fragments containing multiple potential promoters for short RNA synthesis
(“promoter islands”). The method developed here enables one-step purification of H-NS protein using
affinity chromatography and allows investigating efficiency of H-NS binding to the DNA targets by detecting
complexes after specific sorption of anti-his antibodies to the his-tagged protein.