Identification, expression, and crystallization of the protease-resistant conserved domain of synapsin I.
Protein Sci
; 6(10): 2264-7, 1997 Oct.
Article
em En
| MEDLINE
| ID: mdl-9336850
ABSTRACT
A 35-37-kDa protease-resistant domain of synapsin Ia/ Ib, apparently produced by low levels of endogenous proteases in vapor diffusion droplets, slowly formed crystals diffracting X-rays to approximately 10 A resolution. The fragment mainly consisted of the highly conserved C domain common to the synapsin I/II family plus short N- and C-terminal flanking segments. Two constructs (SynA and SynB) of synthetic gene fragments coding for the C domain of synapsin with or without C-terminal flanking sequence were expressed in Escherichia coli as fusion proteins attached to the soluble protein glutathione-S-transferase. The fusion proteins were purified by affinity chromatography. Subsequent in situ cleavage with TEV protease resulted in the release of highly pure synapsin fragments, which were further purified by ion exchange chromatography. SynA and SynB formed crystals within three days, which diffracted to better than 3 A using a conventional X-ray source and to about 2 A using a synchrotron X-ray source. SynA crystals have the symmetry of the trigonal space groups P3(1)21 or P3(2)21 and the unit cell dimensions a = b = 77.4 A, c = 188.5 A, alpha = beta = 90 degrees, gamma = 120 degrees. SynB crystals have the symmetry of the orthorhombic space group C222(1) with the unit cell dimension a = 104.6 A, b = 113.3 A, and c = 273.8 A.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Sinapsinas
Tipo de estudo:
Diagnostic_studies
Idioma:
En
Revista:
Protein Sci
Assunto da revista:
BIOQUIMICA
Ano de publicação:
1997
Tipo de documento:
Article
País de afiliação:
Estados Unidos