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 ∼ 10 Å 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 Å using a conventional X-ray source and to about 2 Å using a synchrotron X-ray source. SynA crystals have the symmetry of the trigonal space groups P3₁21 or P3₂21 and the unit cell dimensions a = b = 77.4 Å, c = 188.5 Å, α = β = 90°, γ = 120°. SynB crystals have the symmetry of the orthorhombic space group C222₁ with the unit cell dimensions a = 104.6 Å, b = 113.3 Å, and c = 273.8 Å. [ABSTRACT FROM AUTHOR]