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.
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Abbreviations
- ATP:
-
Adenosine triphosphate
- BEVS:
-
Baculovirus expression vector system
- COP9:
-
Constitutive photomorphogenesis 9
- DTT:
-
1,4-Dithiothreitol
- GST:
-
Glutathione S-transferase
- IGEPAL:
-
Octylphenoxy poly(ethyleneoxy)etanol
- InsP:
-
Inositol phosphate
- IP5 :
-
Inositol pentakisphosphate
- IP5 2-K:
-
Inositol 1,3,4,5,6-pentakisphosphate 2-kinase
- IP6 :
-
Inositol hexakisphosphate
- IP7 :
-
Diphospho-inositol pentakisphosphate
- IP8 :
-
Bisdiphospho-inositol tetrakisphosphate
- IPK:
-
Inositol polyphosphate kinase
- IPTG:
-
Isopropyl-β-d-1-thiogalactopyranoside
- LSL:
-
Laetiporus sulphurous lectin
- PEG:
-
Polyethylene glycol
- PMSF:
-
Phenylmethanesulfonyl-fluoride
- SDS-PAGE:
-
Sodium dodecyl sulphate polyacrylamide gel electrophoresis
- SF21:
-
Sodopter Frugiperda cell line
- TEV:
-
Tobacco etch virus
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Acknowledgements
We would like to thank Dr. Juana María Gonzalez Rubio for technical assistance and help with preparation in protein constructs. We thank the European Synchrotron Radiation Facility (Grenoble, France) and ALBA CELLS XALOC 13 for providing beam time and assistance during data collection. The authors acknowledge the support and the use of resources of the French Infrastructure for Integrated Structural Biology FRISBI ANR-10-INSB-05 and of Instruct, a Landmark ESFRI project. We would also like to thank the staff of the mass spectrometry facility at the IQFR-CSIC. This work and E.F.-E. were supported by Grants BFU2011-24982 and BFU2014-53762-P from the “Spanish Ministry of Economy and Competitiveness”.
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Franco-Echevarría, E., Sanz-Aparicio, J., Troffer-Charlier, N. et al. Crystallization and Preliminary X-Ray Diffraction Analysis of a Mammal Inositol 1,3,4,5,6-Pentakisphosphate 2-Kinase. Protein J 36, 240–248 (2017). https://doi.org/10.1007/s10930-017-9717-y
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DOI: https://doi.org/10.1007/s10930-017-9717-y