To test whether a recombinant His-tagged version of SPNR produced in E. coli could be purified with testis microtubules, as is the endogenous SPNR protein, testis microtubules were pelleted and resuspended in SPNR-con-taining E. coli extracts prepared as described in Materials and Methods. These microtubule-enriched extracts were subjected to two further rounds of centrifugation and washing, and aliquots from each supernatant and pellet were assayed by SDS-PAGE followed by Coomassie staining or Western blotting. As shown in Figure 6A, the addition of E. coli extracts did not interfere with the purification of tubulin, and taxol was still necessary for the continued enrichment of tubulin through three rounds of purification. Gels duplicate to those shown in Figure 6A were blotted onto nitrocellulose and incubated with an a-His-tag antibody specific for the exogenous E. coli-produced SPNR protein (Fig. 6B). The SPNR protein added to the first pellet remained in the microtubule pellet through two further rounds of purification, an association that was taxol dependent. Thus, the His-tagged SPNR protein behaves in a manner similar to that of the endogenous testicular protein. birth control yasmin
To ascertain whether the association of SPNR with microtubules occurs by direct binding to tubulin, or via a MAP, MAP-free bovine brain tubulin was added to His-tagged SPNR E. coli extracts, and microtubule polymerization was induced by incubating the extract at 37°C in the presence of GTP and taxol. Microtubules were pelleted, washed, and repelleted.
FIG. 6. Exogenous SPNR binds to testis microtubules. A) Microtubules pelleted from testis extracts with and without the addition of taxol were resuspended in His-tagged SPNR-containing E. coli extracts. Two additional rounds of pelleting and washing of the microtubules were performed. Aliquots of the starting testis extract, each supernatant, and each pellet were run on SDS-PAGE gels and stained with Coomassie blue. The E. coli lysate was added to the first pellet. B) Western blots of duplicate gels to those shown in A were probed with an anti-His antibody specific for the His-tagged SPNR protein. Molecular weight standards (X 10~3) are on the left.