Engineering of histidine tail in the N-terminal region of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens

Abstract

The pobA of Pseudomonas florescens IFO14160 encoding a p-hydroxybenzoate hydroxylase (PHBH) was cloned, sequenced, and over-expressed in Escherichia coli. To facilitate the purification of PHBH, a fully active, tagged enzyme was constructed by engineering a two-, three-, or six-histidine tail in the N-terminal region (H2-, H3-, or H6-PHBH), or a six-histidine tail in the C-terminal region of the PHBH. The six-histidine tail in the C-terminal region of the enzyme could not be expressed with activity, while the other polyhistidine tails in the N-terminal region of the enzyme were highly active. However, the resulting H6-PHBH could not be purified by Ni-NTA chromatography because the H6-PHBH was so strongly bound to the supports that it could be eluted only after a significant change in conditions. On the other hand, H2-PHBH did not bind tightly to the Ni-chelate support. H3-PHBH was purified from the crude extract in a single step by using the optimized length and location of the polyhistidine tail in the enzyme.