Identification, expression, and crystallization of the protease-resistant conserved domain of synapsin I.
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.
Wang
C R
CR
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas 75235-9050, USA.
Esser
L
L
Smagula
C S
CS
Südhof
T C
TC
Deisenhofer
J
J
D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry; D011993:Recombinant Fusion Proteins; D016704:Synapsins / Q000737:chemistry* / Q000235:genetics
D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D016704:Synapsins; D010450:Endopeptidases; C099488:TEV protease