Cloning, expression, purification, characterization, crystallization and X-ray crystallographic analysis of recombinant Derf21 (rDerf21) from Dermatophagoides farinae

Sze Lei Pang, Kok Lian Ho, Jitka Waterman, Aik Hong Teh, Fook Tim Chew, Chyan Leong Ng

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

Dermatophagoides farinae is one of the major house dust mite (HDM) species that cause allergic diseases. N-terminally His-tagged recombinant Derf21 (rDerf21), a group 21 allergen, with the signal peptide truncated was successfully overexpressed in an Escherichia coli expression system. The purified rDerf21 protein was initially crystallized using the sitting-drop vapour-diffusion method. Well diffracting protein crystals were obtained after optimization of the crystallization conditions using the hanging-drop vapour-diffusion method with a reservoir solution consisting of 0.19M Tris-HCl pH 8.0, 32% PEG 400 at 293K. X-ray diffraction data were collected to 1.49Å resolution using an in-house X-ray source. The crystal belonged to the C-centered monoclinic space group C2, with unit-cell parameters a = 123.46, b = 27.71, c = 90.25Å, β = 125.84°. The calculated Matthews coefficient (V M) of 2.06Å3Da-1 suggests that there are two molecules per asymmetric unit, with a solvent content of 40.3%. Despite sharing high sequence identity with Blot5 (45%) and Blot21 (41%), both of which were determined to be monomeric in solution, size-exclusion chromatography, static light scattering and self-rotation function analysis indicate that rDerf21 is likely to be a dimeric protein.

Original languageEnglish
Pages (from-to)1396-1400
Number of pages5
JournalActa Crystallographica Section F:Structural Biology Communications
Volume71
DOIs
Publication statusPublished - 2015

Fingerprint

Dermatophagoides farinae
Cloning
Crystallization
purification
Purification
Organism Cloning
Vapors
X-Rays
crystallization
proteins
X rays
Pyroglyphidae
Crystals
allergic diseases
Size exclusion chromatography
Protein Sorting Signals
Recombinant Proteins
X-Ray Diffraction
Light scattering
Allergens

Keywords

  • Dermatophagoides farinae
  • house dust-mite allergen
  • rDer f 21

ASJC Scopus subject areas

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

Cite this

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title = "Cloning, expression, purification, characterization, crystallization and X-ray crystallographic analysis of recombinant Derf21 (rDerf21) from Dermatophagoides farinae",
abstract = "Dermatophagoides farinae is one of the major house dust mite (HDM) species that cause allergic diseases. N-terminally His-tagged recombinant Derf21 (rDerf21), a group 21 allergen, with the signal peptide truncated was successfully overexpressed in an Escherichia coli expression system. The purified rDerf21 protein was initially crystallized using the sitting-drop vapour-diffusion method. Well diffracting protein crystals were obtained after optimization of the crystallization conditions using the hanging-drop vapour-diffusion method with a reservoir solution consisting of 0.19M Tris-HCl pH 8.0, 32{\%} PEG 400 at 293K. X-ray diffraction data were collected to 1.49{\AA} resolution using an in-house X-ray source. The crystal belonged to the C-centered monoclinic space group C2, with unit-cell parameters a = 123.46, b = 27.71, c = 90.25{\AA}, β = 125.84°. The calculated Matthews coefficient (V M) of 2.06{\AA}3Da-1 suggests that there are two molecules per asymmetric unit, with a solvent content of 40.3{\%}. Despite sharing high sequence identity with Blot5 (45{\%}) and Blot21 (41{\%}), both of which were determined to be monomeric in solution, size-exclusion chromatography, static light scattering and self-rotation function analysis indicate that rDerf21 is likely to be a dimeric protein.",
keywords = "Dermatophagoides farinae, house dust-mite allergen, rDer f 21",
author = "Pang, {Sze Lei} and Ho, {Kok Lian} and Jitka Waterman and Teh, {Aik Hong} and Chew, {Fook Tim} and Ng, {Chyan Leong}",
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T1 - Cloning, expression, purification, characterization, crystallization and X-ray crystallographic analysis of recombinant Derf21 (rDerf21) from Dermatophagoides farinae

AU - Pang, Sze Lei

AU - Ho, Kok Lian

AU - Waterman, Jitka

AU - Teh, Aik Hong

AU - Chew, Fook Tim

AU - Ng, Chyan Leong

PY - 2015

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N2 - Dermatophagoides farinae is one of the major house dust mite (HDM) species that cause allergic diseases. N-terminally His-tagged recombinant Derf21 (rDerf21), a group 21 allergen, with the signal peptide truncated was successfully overexpressed in an Escherichia coli expression system. The purified rDerf21 protein was initially crystallized using the sitting-drop vapour-diffusion method. Well diffracting protein crystals were obtained after optimization of the crystallization conditions using the hanging-drop vapour-diffusion method with a reservoir solution consisting of 0.19M Tris-HCl pH 8.0, 32% PEG 400 at 293K. X-ray diffraction data were collected to 1.49Å resolution using an in-house X-ray source. The crystal belonged to the C-centered monoclinic space group C2, with unit-cell parameters a = 123.46, b = 27.71, c = 90.25Å, β = 125.84°. The calculated Matthews coefficient (V M) of 2.06Å3Da-1 suggests that there are two molecules per asymmetric unit, with a solvent content of 40.3%. Despite sharing high sequence identity with Blot5 (45%) and Blot21 (41%), both of which were determined to be monomeric in solution, size-exclusion chromatography, static light scattering and self-rotation function analysis indicate that rDerf21 is likely to be a dimeric protein.

AB - Dermatophagoides farinae is one of the major house dust mite (HDM) species that cause allergic diseases. N-terminally His-tagged recombinant Derf21 (rDerf21), a group 21 allergen, with the signal peptide truncated was successfully overexpressed in an Escherichia coli expression system. The purified rDerf21 protein was initially crystallized using the sitting-drop vapour-diffusion method. Well diffracting protein crystals were obtained after optimization of the crystallization conditions using the hanging-drop vapour-diffusion method with a reservoir solution consisting of 0.19M Tris-HCl pH 8.0, 32% PEG 400 at 293K. X-ray diffraction data were collected to 1.49Å resolution using an in-house X-ray source. The crystal belonged to the C-centered monoclinic space group C2, with unit-cell parameters a = 123.46, b = 27.71, c = 90.25Å, β = 125.84°. The calculated Matthews coefficient (V M) of 2.06Å3Da-1 suggests that there are two molecules per asymmetric unit, with a solvent content of 40.3%. Despite sharing high sequence identity with Blot5 (45%) and Blot21 (41%), both of which were determined to be monomeric in solution, size-exclusion chromatography, static light scattering and self-rotation function analysis indicate that rDerf21 is likely to be a dimeric protein.

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