Functional expression and characterization of human 101F6 protein, a homologue of cytochrome b561 and a candidate tumor suppressor gene product

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 b561 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.

Properties of Human Tumor Suppressor 101F6 Protein as a Cytochrome b561 and Its Preliminary Crystallization Trials

“…Procedure for a molecular cloning of the human 101F6 gene was described previously (Recuenco et al, 2009). Construction of the expression plasmid, pPICZB-101F6-His 8 , was described previously (Recuenco et al, 2009).…”

“…Construction of the expression plasmid, pPICZB-101F6-His 8 , was described previously (Recuenco et al, 2009). Briefly, the thrombin-specific sequence followed by the eight-histidine residue-tag (QPSALVPRGSSAHHHHHHHH; the underline indicates the thrombin-specific sequence) was introduced at the C-terminus of human 101F6 protein, resulting in a total of 240 aa residue-long with a molecular mass of 25996.9 Da.…”

“…Single colonies with a hyper Zeocin-resistant activity from the YPDS-Zeocin plates were inoculated into 1000-mL Erlenmeyer flasks with a baffled bottom containing 250 mL BMGY media (2% glycerol). During the usual induction procedure of such transformants by the addition of methanol (final 2%) as described previously (Recuenco et al, 2009), the color of the medium become reddish, indicating a successful expression of a holo-form of the 101F6 protein in the Pichia pastoris cells. After harvesting the induced cells, microsomal fractions were prepared in the presence of protease inhibitor cocktail (Protease Inhibitor Cocktail for General Use (100X); Nacalai Tesque, Kyoto, Japan).…”

“…However, instances about its use as a host for the expression of mammalian membrane proteins was limited (Long et al, 2005). We have previously reported about the functional expression, purification and characterization of recombinant human 101F6 protein, which was expressed as a polyhistidine tagged protein in methylotrophic yeast Pichia pastoris (Recuenco et al, 2009). The purified 101F6 protein exhibited characteristic properties as a member of the cytochrome b 561 protein family, particularly with regards to spectral characteristics and electron transfer activities with AsA and MDA radical.…”

Dihydrolipoic acid reduces cytochrome b561 proteins

“…It seems that other members of the protein family are present in a very small amount in their natural sources and appropriate amounts of these Cyt-b561 proteins could be obtained only after establishing a heterologous expression system for producing recombinant Cyt-b561 proteins. Such expression systems were developed and successfully used in the characterization of CGCB (Bérczi et al 2005;Liu et al 2005Liu et al , 2007, of the duodenum-localized Cyt-b561 (DCB; Ludwiczek et al 2008;Oakhill et al 2008), of the lysosome-localized Cyt-b561 (LCB; Zhang et al 2006), of the putative tumour suppressor Cytb561 (TSCB; Mizutani et al 2007;Bérczi and Asard 2008;Recuenco et al 2009), and of the tonoplast-localized Cyt-b561 (TCB; Griesen et al 2004;Bérczi et al 2007;Cenacchi et al 2011) in the last 10 years. Only speculations exist, however, for the biological function of Cyt-b561 proteins other than CGCB and DCB (e.g.…”