title: Cloning, expression, purification, characterization, crystallization and X-ray crystallographic analysis of recombinant Der f 21 (rDer f 21) 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 Der f 21 (rDer f 21), a group 21 allergen, with the signal peptide truncated was successfully overexpressed in an Escherichia coli expression system. The purified rDer f 21 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 0.19 M Tris-HCl pH 8.0, 32% PEG 400 at 293 K. X-ray diffraction data were collected to 1.49 Å 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 Å, β = 125.84°. The calculated Matthews coefficient (VM) of 2.06 Å(3) Da(-1) suggests that there are two molecules per asymmetric unit, with a solvent content of 40.3%. Despite sharing high sequence identity with Blo t 5 (45%) and Blo t 21 (41%), both of which were determined to be monomeric in solution, size-exclusion chromatography, static light scattering and self-rotation function analysis indicate that rDer f 21 is likely to be a dimeric protein.
authors.affiliation: Institute of Systems Biology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia.
authors.forename: Sze Lei
authors.initials: SL
authors.lastname: Pang
authors.affiliation: Department of Pathology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia.
authors.forename: Kok Lian
authors.initials: KL
authors.lastname: Ho
authors.affiliation: Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE, England.
authors.forename: Jitka
authors.initials: J
authors.lastname: Waterman
authors.affiliation: Centre for Chemical Biology, Universiti Sains Malaysia, 10 Persiaran Bukit Jambul, 11900 Bayan Lepas, Penang, Malaysia.
authors.forename: Aik Hong
authors.initials: AH
authors.lastname: Teh
authors.affiliation: Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543, Singapore.
authors.forename: Fook Tim
authors.initials: FT
authors.lastname: Chew
authors.affiliation: Institute of Systems Biology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia.
authors.forename: Chyan Leong
authors.initials: CL
authors.lastname: Ng
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D039741:Antigens, Dermatophagoides / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D060829:Arthropod Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D003001:Cloning, Molecular / Q000379:methods*; D003460:Crystallization; D018360:Crystallography, X-Ray; D040001:Dermatophagoides farinae / Q000235:genetics*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D039741:Antigens, Dermatophagoides; D060829:Arthropod Proteins; D011994:Recombinant Proteins
keywords: Dermatophagoides farinae; house dust-mite allergen; rDer f 21