Lupanine hydroxylase (LH) is a quinohaemoprotein responsible for the conversion of the 25 alkaloid, lupanine to 17-hydroxylupanine. Previous attempts to express the enzyme in 26 Escherichia coli required in vitro addition of the co-factor pyrroloquinoline quinone 27 (PQQ) and posed some impediments on subsequent structural studies for further 28 characterization of the enzyme. An E. coli clone with LH and cytochrome c maturation 29 operon was transformed with a third plasmid containing the PQQ operon from Klebsiella 30 pneumoniae , luh gene and resulted in the production of periplasmically-targeted, 31 correctly folded, PQQ and haem inserted active enzyme. 32 Interestingly, LH was less active than the in vitro incorporated PQQ-LH, presumably due 33 to the incorporation of PQQ precursors in the periplasm. This is a first report of an active 34 LH enzyme with in vivo incorporation of PQQ in E. coli and provides the necessary tool 35 for further enzyme structural characterization.