Cloning, expression, purification, characterization, crystallization and X-ray crystallographic analysis of recombinant Der f 21 (rDer f 21) from Dermatophagoides farinae

DOI: 10.1107/S2053230X1501818X
PMID: 26527267

Authors: Sze Lei Pang (Universiti Kebangsaan Malaysia) , Kok Lian Ho (Putra University, Malaysia) , Jitka Waterman (Diamond Light Source) , Aik-Hong Teh (Universiti Sains Malaysia) , Fook Tim Chew (National University of Singapore) , Chyan Leong Ng (National University of Malaysia)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Acta Crystallographica Section F Structural Biology Communications , VOL 71 , PAGES 1396 - 1400

State: Published (Approved)
Published: November 2015

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, [beta] = 125.84°. The calculated Matthews coefficient (VM) of 2.06 Å3 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.

Journal Keywords: Dermatophagoides Farinae; Rder F 21; House Dust-Mite Allergen.

Subject Areas: Biology and Bio-materials


Technical Areas:

Added On: 11/12/2015 12:11

Documents:
us5084.pdf

Discipline Tags:

Non-Communicable Diseases Health & Wellbeing Structural biology Life Sciences & Biotech Allergic Diseases

Technical Tags: