Gel electrophoretic isolation, in the hundred microgram range, of recombinant SDS-syntaxin from sea urchin egg cortical vesicles

Recombinant urchin syntaxin [Xa cut], electrophoresed at pH 9.0 (25 degrees C) or 10.2 (0 degreesC) in a discontinuous Tris-chloride-glycinate buffer s ystem in the presence of 0.03% SDS in the catholyte, exhibits a multicompon ent pattern in gels of a polyacrylamide concentration of 12% and 3% crossli nking. The position in the pattern of the syntaxin band was identified by r eference to electropherograms of a previous study (P. Backlund, pers. comm. ). The complexity of the protein composition of the preparation was reduced by selective stacking of proteins with mobilities greater than that of syn taxin. This provides a gel pattern consisting of two bands with mobilities close to that identified as syntaxin, as well as a minor, more slowly migra ting, contaminant. The two major components are designated as S1 and S2, th e latter being the larger species. In the absence of SDS, the preparation e xhibits two pairs of protein components. Three of the proteins are charge i somers, i.e., of equal size, differing only in net charge, assumed to be fo rms of S1, while the fourth component is larger and is assumed to be S2. Aliquots of the preparation, containing 150 mug of protein were loaded on a cylindrical polyacrylamide gel of 18 mm. diameter, and separated S1 and S2 were excised in a position defined by their characteristic values of relat ive mobility (R-f). Two or three gel slices, corresponding in R-f to S1 or S2, were pooled and loaded onto a Stacking Gel (5% polyacrylamide, 20% cros slinked) of 18 mm diameter, equipped with a collection chamber of 200 muL v olume. The protein was electroeluted from the gel slices and concentrated i nto a stack by electrophoresis. The stack, marked by bromphenolblue, was al lowed to migrate into the collection chamber, was collected and analyzed by protein assay and re-electrophoresis. Re-electrophoresis of S1 shows that it consists of at least three components. Recovered S1 constitutes 47% of t he preparation, based on protein assay, S2 4%. S1, isolated from SDS-PAGE, exhibits an apparent M-r of 22.7 kDa, S2 one of 34.5 kDa, similar to the va lue of 32.6 kDa expected from the structure of syntaxin. The absence of S2 from the electroeluate re-electrophoresed at 0 degrees C and their molecula r weight relationship suggest a proteolytic transformation of S2 to S1.