Nisin biosynthesis protein NisB, encoded by the nisB gene, is a membrane-associated enzyme of 993 amino acid residues which carries out the selective dehydration of the serine and threonine residues of the precursor nisin, leading to dehydroalanine (Dha) and dehydrobutyrine (Dhb), respectively. In this study, the nisB gene of Lactococcus lactis FI5876 was cloned into the expression vector pET-15b, under the control of the isopropyl beta-D-1-thiogalactopyranoside (IPTG)-inducible T7 promoter, expressed as an N-terminal His(6)-tag fusion protein in Escherichia coli, and purified by nickel-affinity chromatography. The identity of the recombinant NisB protein was confirmed by western blot analysis using antibodies specific for NisB or the His(6)-tag. The circular dichroism spectrum of His(6)-tagged NisB was also obtained, which provided secondary structure information. We report here the heterologous expression of the nisB gene of a nisin A-producing Lactococcus lactis strain in Escherichia coli and purification of His(6)-tagged NisB under native conditions.