Purification, crystallization, and X-ray diffraction analysis of myocyte enhancer factor 2D and DNA complex.
Protein Expr Purif
; 179: 105788, 2021 03.
Article
en En
| MEDLINE
| ID: mdl-33221504
ABSTRACT
MEF2D-fusions have recently been identified as one of the major oncogenic drivers in precursor B-cell acute lymphoblastic leukemia (B-ALL). More importantly, they are often associated with patients with poor prognosis in B-ALL. To have a better understanding of the pathogenic mechanism underpinning MEF2D-fusions-driven leukemogenesis, it's essential to uncover the related structure information. In this study, we expressed and purified the MEF2D N-terminal DNA binding domain. The recombinant protein was engineered by cloning the encoding gene into the expression vector pET-32 m. A series of chromatographic steps involving affinity, ion-exchange and gel-filtration chromatography were used to achieve a final purity of >95%. For the crystallization of the MEF2D-DNA complex, a double-stranded DNA encoding 5'-AACTATTTATAAGA-3' and 5'-TTCTTATAAATAGT-3' was used (Wu et al., 2010) [1]. The MEF2D-DNA crystal with the size of about 20 µm × 20 µm × 20 µm was obtained at a final concentration of 12 mg/ml at the reservoir condition containing 30% PEG1500. The X-ray examination showed that the MEF2D-DNA crystal diffracted to 4.5 Å resolution, and belonged to space group P1, with unit-cell parameters of a = 77.2 Å, b = 77.2 Å, c = 231.4 Å.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Proteínas Recombinantes
/
ADN
Tipo de estudio:
Prognostic_studies
Límite:
Humans
Idioma:
En
Revista:
Protein Expr Purif
Asunto de la revista:
BIOLOGIA MOLECULAR
Año:
2021
Tipo del documento:
Article