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Cloning, expression, purification, characterization, crystallization and X‐ray crystallographic analysis of recombinant Der f 21 (rDer f 21) from Dermatophagoides farinae
Author(s) -
Pang Sze Lei,
Ho Kok Lian,
Waterman Jitka,
Teh AikHong,
Chew Fook Tim,
Ng Chyan Leong
Publication year - 2015
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 37
ISSN - 2053-230X
DOI - 10.1107/s2053230x1501818x
Subject(s) - crystallization , recombinant dna , chemistry , crystallography , circular dichroism , monoclinic crystal system , escherichia coli , chromatography , crystal structure , biochemistry , organic chemistry , gene
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 C 2, with unit‐cell parameters a = 123.46, b = 27.71, c = 90.25 Å, β = 125.84°. The calculated Matthews coefficient ( V M ) 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.