Abstract
C1ORF123 is a human hypothetical protein found in open reading frame 123 of chromosome 1. The protein belongs to the DUF866 protein family comprising eukaryote-conserved proteins with unknown function. Recent proteomic and bioinformatic analyses identified the presence of C1ORF123 in brain, frontal cortex and synapses, as well as its involvement in endocrine function and polycystic ovary syndrome (PCOS), indicating the importance of its biological role. In order to provide a better understanding of the biological function of the human C1ORF123 protein, the characterization and analysis of recombinant C1ORF123 (rC1ORF123), including overexpression and purification, verification by mass spectrometry and a Western blot using anti-C1ORF123 antibodies, crystallization and X-ray diffraction analysis of the protein crystals, are reported here. The rC1ORF123 protein was crystallized by the hanging-drop vapor-diffusion method with a reservoir solution comprised of 20% PEG 3350, 0.2 M magnesium chloride hexahydrate, 0.1 M sodium citrate pH 6.5. The crystals diffracted to 1.9 Å resolution and belonged to an orthorhombic space group with unit-cell parameters a = 59.32, b = 65.35, c = 95.05 Å. The calculated Matthews coefficient (VM) value of 2.27 Å(3) Da(-1) suggests that there are two molecules per asymmetric unit, with an estimated solvent content of 45.7%.
Highlights
Open reading frame 123, which is located in the short arm of human chromosome 1, encodes a hypothetical protein known as C1ORF123 (Selvarajan & Shanmughavel, 2014)
Other proteomics studies have found that the C1ORF123 protein is largely expressed in the hippocampus of people suffering from schizophrenia, bipolar disorder and methamphetamine-induced sensitization of the prefrontal cortex, as well as being a unique protein in the frontal cortex of aged rats associated with slowwave sleep (SWS) (Schubert et al, 2015; Wearne et al, 2015; Vazquez et al, 2009)
The concentrated rC1ORF123 protein was further purified by size-exclusion chromatography (SEC) using a HiLoad 16/600 Superdex 75 pg gel-filtration column (GE Healthcare, USA) pre-equilibrated with size-exclusion buffer consisting of 25 mM Tris–HCl pH 7.5, 100 mM NaCl, 20 mM -mercaptoethanol
Summary
Open reading frame 123, which is located in the short arm of human chromosome 1, encodes a hypothetical protein known as C1ORF123 (Selvarajan & Shanmughavel, 2014). Other proteomics studies have found that the C1ORF123 protein is largely expressed in the hippocampus of people suffering from schizophrenia, bipolar disorder and methamphetamine-induced sensitization of the prefrontal cortex, as well as being a unique protein in the frontal cortex of aged rats associated with slowwave sleep (SWS) (Schubert et al, 2015; Wearne et al, 2015; Vazquez et al, 2009). This indicates the involvement of C1ORF123 in psychotic diseases or in age-related changes in brain function. We report the cloning, overexpression, purification, protein characterization and crystallization together with the initial X-ray crystallographic analysis of recombinant C1ORF123 (rC1ORF123)
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