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Functional expression and characterization of human 101 F 6 protein, a homologue of cytochrome b 561 and a candidate tumor suppressor gene product
Author(s) -
Recuenco Mariam C.,
Fujito Masamitsu,
Sakamoto Yoichi,
Tsubaki Motonari,
Rahman M. D. Motiur,
Takeuchi Fusako
Publication year - 2008
Publication title -
biofactors
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.204
H-Index - 94
eISSN - 1872-8081
pISSN - 0951-6433
DOI - 10.1002/biof.5520340306
Subject(s) - pichia pastoris , biochemistry , microbiology and biotechnology , hspa2 , biology , affinity chromatography , cytochrome , fusion protein , protein a/g , transmembrane domain , vesicle associated membrane protein 8 , transmembrane protein , peptide sequence , retinoblastoma like protein 1 , chemistry , gene , membrane protein , recombinant dna , enzyme , membrane , receptor
A highly hydrophobic protein with six transmembrane structure that is coded by the candidate tumor suppressor gene 101 F 6 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 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.