Cloning, expression, purification, crystallization and X-ray crystallographic analysis of recombinant human C1ORF123 protein

Siti Nurulnabila A Rahaman, Jastina Mat Yusop, Zeti Azura Mohamed Hussein, Kok Lian Ho, Aik Hong Teh, Jitka Waterman, Chyan Leong Ng

Research output: Contribution to journalArticle

1 Citation (Scopus)

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% resolution and belonged to an orthorhombic space group with unit-cell parameters a = 59.32, b = 65.35, c = 95.05%. The calculated Matthews coefficient (V M) value of 2.27% A3 Da-1 suggests that there are two molecules per asymmetric unit, with an estimated solvent content of 45.7%.

Original languageEnglish
Pages (from-to)207-213
Number of pages7
JournalActa Crystallographica Section F:Structural Biology Communications
Volume72
DOIs
Publication statusPublished - 2016

Fingerprint

Cloning
Crystallization
purification
Purification
Organism Cloning
X-Rays
crystallization
proteins
X rays
Proteins
x rays
Crystals
Magnesium Chloride
eukaryotes
magnesium chlorides
Polycystic Ovary Syndrome
Chromosomes, Human, Pair 1
synapses
Frontal Lobe
ovaries

Keywords

  • bioinformatic analysis
  • C1ORF123
  • DUF866
  • hypothetical protein
  • polycystic ovary syndrome

ASJC Scopus subject areas

  • Biophysics
  • Genetics
  • Structural Biology
  • Condensed Matter Physics
  • Biochemistry

Cite this

Cloning, expression, purification, crystallization and X-ray crystallographic analysis of recombinant human C1ORF123 protein. / Rahaman, Siti Nurulnabila A; Yusop, Jastina Mat; Mohamed Hussein, Zeti Azura; Ho, Kok Lian; Teh, Aik Hong; Waterman, Jitka; Ng, Chyan Leong.

In: Acta Crystallographica Section F:Structural Biology Communications, Vol. 72, 2016, p. 207-213.

Research output: Contribution to journalArticle

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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{\%} resolution and belonged to an orthorhombic space group with unit-cell parameters a = 59.32, b = 65.35, c = 95.05{\%}. The calculated Matthews coefficient (V M) value of 2.27{\%} A3 Da-1 suggests that there are two molecules per asymmetric unit, with an estimated solvent content of 45.7{\%}.",
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AB - 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% resolution and belonged to an orthorhombic space group with unit-cell parameters a = 59.32, b = 65.35, c = 95.05%. The calculated Matthews coefficient (V M) value of 2.27% A3 Da-1 suggests that there are two molecules per asymmetric unit, with an estimated solvent content of 45.7%.

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