Affiliations: Department of Chemistry, Graduate School of Science,
Kobe University, Kobe, Hyogo, Japan | Department of Molecular Science, Graduate School of
Science and Technology, Kobe University, Kobe, Hyogo, Japan | Department of Pharmacy, College of Pharmaceutical
Sciences, Ritsumeikan University, Kusatsu, Shiga, Japan
Note: [] Address for correspondence: Motonari Tsubaki, Department of
Chemistry, Graduate School of Science, Kobe University, 1-1 Rokkodai-cho,
Nada-ku, Kobe, Hyogo 657-8501, Japan. Tel./Fax: +81 78 803 6582; E-mail:
[email protected]
Abstract: A highly hydrophobic protein with six transmembrane structure that
is coded by the candidate tumor suppressor gene 101F6
located in the human chromosome 3p.21.3 and a possible member of the cytochrome
b_{561} protein family was expressed, purified, and
characterized in its functional form for the first time. The protein was
heterologously expressed in methylotrophic yeast Pichia pastoris as a
fusion protein containing a C-terminal thrombin-specific sequence and an 8-His
residue tag. Purification was achieved by ion exchange chromatography on
DEAE-Sepharose and affinity chromatography on Ni-NTA-Sepharose. SDS-PAGE
analysis revealed a single protein band with an estimated molecular weight of
26 kDa, while Western blot and MALDI-TOF-MS analysis confirmed the presence of
the cytochrome b_{561}-specific sequence in the protein. The
UV-Visible spectra of the protein in the reduced and oxidized states showed the
characteristic peaks (561, 528, 427 nm for the reduced form and 414 nm for the
oxidized form) typical of cytochrome b_{561} proteins. The
101F6 protein was found to be reducible by ascorbate efficiently and to have
two midpoint potentials at +89.5 and
+13.1 mV, slightly lower than the corresponding values of
+155 and +62 mV, respectively, of bovine
adrenal cytochrome b_{561}, despite a lower conservation of
the putative ascorbate binding site sequence in the 101F6 protein. The
"modified motif 1" sequence unique in 101F6 protein may be responsible for
other molecular functions, such as protein-protein interactions, in the
endoplasmic membranes.
DOI: 10.3233/BIO-2009-1075
Journal: BioFactors, vol. 34, no. 3, pp. 219-230, 2009
