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Purified promyelocytic leukemia coiled-coil aggregates as a tetramer displaying low alpha-helical content.

Abstract：

:The promyelocytic leukemia (PML) gene is involved in the 15/17 chromosomal translocation of acute promyelocytic leukemia (APL). It encodes a nuclear phosphoprotein containing an alpha-helical coiled-coil domain with four heptad repeats. The heptad repeats consist of four clusters of hydrophobic amino acids that mediate in vivo the complex formation between PML and other PML molecules or PML-RARalpha mutant protein. In this report, we show the production of PML coiled-coil (fragment 223-360) as a fusion protein, its solubilization by the combined action of two different detergents, and its purification with affinity chromatography after column proteolytic cleavage. The FPLC chromatograms of the purified coiled-coils, carried out under non-denaturing conditions, show that the peptide elutes only in the presence of Sarkosyl detergent (conc. 0.1%) and, under these conditions, elutes as a tetrameric complex. This confirms the evidence from in vivo experiments that this region is responsible for protein complex formation. The HPLC analyses show the presence of a single peak eluting under highly hydrophobic conditions, indicating the high hydrophobicity of the peptide in accordance with the primary sequence analysis. Finally, the purified peptide was structurally characterized by means of circular dichroism (CD) measurements that were carried out with low Sarkosyl concentration (0.003%). The CD spectra indicate a low alpha-helical content (13.5%) with respect to predictions based on the primary sequence analysis (PSI-PRED, SS-PRO, and J-PRED), suggesting that the alpha-helix content could be modulated by coiled-coil surrounding domains and/or by other post-translational modifications, even if the effect of the Sarkosyl on the peptide secondary structure cannot be excluded.

journal_name

Protein Expr Purif

journal_title

Protein expression and purification

authors

Antolini F,Lo Bello M,Sette M

doi

10.1016/s1046-5928(03)00004-4

keywords:

subject

Has Abstract

pub_date

2003-05-01 00:00:00

pages

94-102

issue

1

eissn

1046-5928

issn

1096-0279

pii

S1046592803000044

journal_volume

29

pub_type

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