Open AccessPurification and Characterization of the Periplasmic Nickel‐Binding Protein NikA of Escherichia coli K12
Author(s) -
Pina Karinne,
Navarro Clarisse,
Mcwalter Laura,
Boxer David H.,
Price Nicholas C.,
Kelly Sharon M.,
MandrandBerthelot MarieAndrée,
Wu LongFei
Publication year - 1995
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1995.0857p.x
Subject(s) - periplasmic space , operon , nickel , escherichia coli , chemistry , biochemistry , binding protein , biology , gene , organic chemistry
The nik operon of Escherichia coli encodes a periplasmic binding‐protein‐dependent transport system specific for nickel. In this report, we describe the overproduction of the periplasmic nickel‐binding protein NikA by cloning the nikA gene into an overexpression vector, pRE1. NikA was purified free of nickel to near homogeneity from the periplasm by hydrophobic and ion‐exchange chromatography. N‐terminal amino acid sequencing confirmed that the leader peptide of NikA had been removed. The nickel‐binding properties of the protein has been studied by monitoring the quenching of intrinsic protein fluorescence. NikA binds one atom of nickel/molecule of protein with a dissociation constant ( K d ) of less than 0.1 μM. Other metals (cobalt, copper, iron) are bound at least 10‐fold less tightly. The high specificity for Ni 2+ is also demonstrated by high‐performance immobilized‐metal‐ion affinity chromatography. Biosynthesis of NikA occurred only under anaerobic conditions and was dependent on the general anaerobic regulator FNR. It was repressed by the presence of 250 μM Ni 2+ in the growth medium and was not affected by either 30 mM formate or 100 mM nitrate. Anaerobically grown wild‐type strain MC4100 contains about 23 000 molecules of NikA/cell. In addition to the effect on nickel transport, nikA mutation affects also the nickel sensing in Tar‐dependent repellent chemotaxis.