Study On The Structure And Function Of SPATA4 Gene Promoter And Purification Of The SPATA4 Protein

Using technique of nested PCR and draft human genome searching, SPATA4 was cloned from a human testis cDNA library. Results of previous research confirmed that SPATA4 was specially expressed in testis of mammalian, and plays an important role in the process of spermatogenic cells apoptosis. Here we study on the structure and function of the SPATA4 gene promoter, and also purification of the SPATA4 protein.Based on the Bioinformatics forecasting, we have established by SMART RACE analysis the transcription start site (TSS) of SPATA4 gene is located in 23bp upstream ATG that corresponds to the first codon of a 305 amino acid protein. This is foundational for researching functionality of transcriptional regulation of promoters.Then, we cloned 2838 bp of the SPATA4 5′-flanking region from a mouse genomic DNA. And seven progressively deleted fragments were cloned in the promoter-less luciferase and the resulting plasmid pGL3-P2838 as the template. After transfected in HeLa cells, we analyzed various 5′-deletions of the promoter region by reporter gene assays. The results indicated that pGL3-P923 had the highest activity. So we suspect the P923 has the core fragment of SPATA4 gene promoter. In addition, bioinformatics comparison showed that there are several transcription factor (TF) binding sites exited in upstream of SPATA4 gene, such as HSF1, RREB1, ZF5. Those TFs are likely to significantly impact the activity of SPATA4 gene promoter.To expression the SPATA4 protein, we cloned the SPATA4 gene to the plasmid pTHM, which added a Maltose Binding Protein (MBP) to the N-terminal of SPATA4. The MBP-tag helps to improve the solubility of SPATA4. The recombination protein, about 80 kD, was preliminary purified by affinity chromatography, and then purified by Ion Exchange Chromatography (IEC) and gel filtration chromatography. Protein harvested last show high purity (about 95%). It could not only be used to produce antibodies, but also helpful for researching on the crystal structure of the protein.