SPAG4L, a novel nuclear envelope protein involved in the meiotic stage of spermatogenesis.

SUN domain-containing proteins belong to a novel protein family. To date, several members--SUN1, SUN2, SUN3, and SPAG4--have been identified as nuclear envelope (NE) proteins. In this study, we sought to characterize and define the potential function of SPAG4L, a newly identified SUN protein. Using bioinformatic analysis, we found that SPAG4L contained a conserved SUN domain in the C-terminal. Subcellular localization analysis indicated that the expression of green fluorescent protein-labeled full-length SPAG4L was localized to the NE and the endoplasmic reticulum (ER). Deletion analysis revealed that the transmembrane region and the coiled-coil domain, but not the SUN domain, were required for localization of SPAG4L to the NE and ER. Subsequently, we confirmed that the human testes expressed endogenous SPAG4L as a 43-kDa protein. Further studies revealed that mouse Spag4L colocalized with the NE marker Lamin B1 and the ER marker PDI in isolated mouse spermatocytes. In addition, the expression of Spag4L was observed in meiosis I and II stages, suggesting that Spag4L may be involved in NE reconstitution and nuclear migration occurring during the process of spermatocyte division. Together, the findings indicate that SPAG4L, a new NE protein, may play an important role in the meiotic stage of spermatogenesis.

[Prokaryotic expression and purification of SPAG4L, a novel human testis gene].

OBJECTIVE: To express SPAG4L, a novel human testis gene in E. coli and purify it's fusion protein. METHODS: The fragment encoding SPAG4L126-379 was amplified by RT-PCR and the PCR products were cloned into PUCm-T vectors. After digestion by EcoR I and Hind III, the fragment was subcloned into PQE-30, a prokaryotic expression vector with 6×His tag. The recombinant plasmid PQE-30-SPAG4L was sequenced and transformed into E.coli M15. The expression of his-tagged fusion protein was induced by IPTG. The fusion protein was identified by Western blotting and purified using Ni-NTA magnetic agarose beads. RESULTS: The recombinant plasmid PQE-30-SPAG4L was constructed successfully and expressed in E.coli M15. The fusion protein SPAG4Lwith 6×his-tag was confirmed by Western blotting. The micro-scale purification system of 6×His-tagged SPAG4Lprotein was established and purified fusion protein was obtained. CONCLUSION: The recombinant plasmid PQE-30-SPAG4L can be expressed in vitro and used for studying the biological function of SPAG4L in spermatogenesis.