Purification and properties of the mercuric-ion-binding protein MerP

Authors


Correspondence to L. Sahlman, Department of Biochemistry, University of Umeå, S-90187 Umeå, Sweden

Abstract

The gene merP, coding for a mercuric-ion-binding periplasmic protein (P protein), was cloned into the expression vector pCA. In an Escherichia coli strain bearing the resulting plasmid, the P protein constitutes about 20% of total soluble protein. P protein was purified using ammonium sulfate precipitation and two chromatography steps. Typical yields were 20–30 mg from 7.5 l bacterial culture. The protein is a monomer with a molecular mass of 7500 Da. The periplasmic signal peptide was processed identically in both the recombinant and the wild-type proteins. CD spectra of both proteins were identical and indicated that the structure is highly ordered, containing approximately 80%α-helix.

Purification in the presence of excess cysteine resulted in a form of the protein containing two reduced thiols, in agreement with the published sequence which has two cysteine residues. When cysteine was omitted from the purification buffers, no reduced thiol groups could be detected suggesting that the cysteine residues are oxidized. Both of these forms of the protein were found to bind approximately five Hg2+ ions/protein molecule in an apparently non-specific manner. However, in the presence of external thiol compounds, the protein with reduced thiols bound only one Hg2+ ion/protein molecule with an apparent Kd of 3.7 ± 1.3 μM. Under these conditions, the protein with oxidized thiols did not bind Hg2+. The possible physiological role of this protein in Hg2+ detoxification is discussed.

Abbreviations
Nbs2

5,5′-dithiobis-(2-nitrobenzoic acid)

protein P and MerP

mercuric-ion-binding protein

Enzymes
 

Mercuric reductase (EC 1.16.1.1)

 

deoxyribonuclease I, DNase (EC 3.1.21.1)

 

ribonuclease A, RNase (EC 3.1.27.5)

 

carbonic anhydrase (EC 4.2.1.1)

 

trypsin (EC 3.4.21.4)

 

T7 RNA polymerase (EC 2.7.7.7)

 

restriction endonucleases (EC 3.1.21.4)

Ancillary