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The coiled‐coil domain of glycosomal membrane‐associated Leishmania donovani PEX14: cloning, overexpression, purification and preliminary crystallographic analysis
Author(s) -
Shakya Anil Kumar,
Pratap J. Venkatesh
Publication year - 2020
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/s2053230x20011127
Subject(s) - cleavage (geology) , chemistry , crystallography , coiled coil , transmembrane protein , transmembrane domain , membrane , biophysics , biology , biochemistry , receptor , paleontology , fracture (geology)
The glycosomal membrane‐associated Leishmania donovani protein PEX14, which plays a crucial role in protein import from the cytosol to the glycosomal matrix, consists of three domains: an N‐terminal domain where the signalling molecule binds, a transmembrane domain and an 84‐residue coiled‐coil domain (CC) that is responsible for oligomerization. CCs are versatile domains that participate in a variety of functions including supramolecular assembly, cellular signalling and transport. Recombinant PEX14 CC was cloned, overexpressed, affinity‐purified with in‐column thrombin cleavage and further purified by size‐exclusion chromatography. Crystals that diffracted to 1.98 Å resolution were obtained from a condition consisting of 1.4 M sodium citrate tribasic dihydrate, 0.1 M HEPES buffer pH 7.5. The crystals belonged to the monoclinic space group C 2, with unit‐cell parameters a = 143.98, b = 32.62, c = 95.62 Å, β = 94.68°. Structure determination and characterization are in progress.