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Expression, Folding and Characterization of C‐Terminally Histidine‐Tagged Styrene Monooxygenase Reductase (SMOB)
Author(s) -
Nallamothu Nagamani,
Chin Franklin,
Kantz Auric,
Gassner George
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.a892
Subject(s) - histidine , biochemistry , inclusion bodies , reductase , chemistry , styrene , gene , biology , amino acid , enzyme , escherichia coli , copolymer , organic chemistry , polymer
In Pseudomonas bacteria styrene is first transformed to styrene oxide by the two‐component flavoenzyme, Styrene Monooxygenase (SMO), which is composed of an NADH‐dependent Flavin reductase (SMOB), and FAD‐dependent Styrene epoxidase (SMOA). Overexpression of native SMOB results predominantly in the formation of inclusion bodies and for this reason recovering a high concentration of both the native and N‐terminally histidine‐tagged versions of this protein has proven to be challenging. Here we report the construction and characteristics of a C‐terminally histidine‐tagged version of SMOB. The styB gene encoding native SMOB was modified through the addition of an Nde 1 site at its 3′ end, and by replacement of the stop codon at the 5′ end with a Kpn1 site. In this way it was possible to insert styB into pET29b with 3′ end of the cloned gene directly in front of nucleotide sequences encoding the C‐terminal Thrombin cleavage site and histidine tag in this vector. Post‐expression SDS‐PAGE analysis reveals that like the native protein, over‐expressed C‐terminally histidine‐tagged SMOB accumulates predominantly in the form of inclusion bodies. After solubilization of the inclusion bodies in urea under reducing conditions, it was possible to recover and rapidly fold the protein into active C‐terminally tagged SMOB. Nickel affinity and ion exchange chromatographies were used for the further purification and recovery of highly active protein. Comparative kinetic studies of the native, N‐terminally, and C‐terminally histidine tagged versions of SMOB will be presented. This work was supported by NIH GM052588.