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Protein J. 2017 Aug;36(4):240-248. doi: 10.1007/s10930-017-9717-y.

Crystallization and Preliminary X-Ray Diffraction Analysis of a Mammal Inositol 1,3,4,5,6-Pentakisphosphate 2-Kinase.

Author information

1
Departament of Crystallography and Structural Biology, Insitute of Physical-Chemistry "Rocasolano," CSIC, Serrano 119, 28006, Madrid, Spain.
2
Institut de Génétique et de Biologie Moléculaire et Cellulaire, UMR 7104, 1 rue Laurent Fries, BP 10142, 67404, Illkirch Cedex, France.
3
Departament of Crystallography and Structural Biology, Insitute of Physical-Chemistry "Rocasolano," CSIC, Serrano 119, 28006, Madrid, Spain. xbeatriz@iqfr.csic.es.

Abstract

Inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IP5 2-K) is an enzyme that catalyses the formation of phytic acid (IP6) from IP5 and ATP. In mammals, IP6 is involved in multiple events such as DNA repair and mRNA edit and it is the precursor of inositol pyrophosphates, emerging compounds shown to have an essential role in apoptosis. In addition, IP5 2-K have functions in cells independently of its catalytic activity, for example in rRNA biogenesis. We pursue the structure determination of a mammal IP5 2-K by Protein Crystallography. For this purpose, we have designed protocols for recombinant expression and purification of Mus musculus IP5 2-K (mIP5 2-K). The recombinant protein has been expressed in two different hosts, E. coli and insect cells using the LSLt and GST fusion proteins, respectively. Both macromolecule preparations yielded crystals of similar quality. Best crystals diffracted to 4.3 Å (E. coli expression) and 4.0 Å (insect cells expression) maximum resolution. Both type of crystals belong to space group P212121 with an estimated solvent content compatible with the presence of two molecules per asymmetric unit. Gel filtration experiments are in agreement with this enzyme being a monomer. Crystallographic data analysis is currently undergoing.

KEYWORDS:

IP5 2-K; IP6; Inositol hexakisphosphate; Inositol kinase; Mammal IPK; X-ray crystallography

PMID:
28429156
DOI:
10.1007/s10930-017-9717-y
[Indexed for MEDLINE]

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