Protein Expression And Purification, Crystal Analysis And Activity

Spermatogenesis is a complex and continuative progress, which was regulated by elaborate mechamism that is the differentially expressed genes. So the identification and study of differentially expressed genes are very important to delineate the regulating spermatogenesis mechanisms and will make great contribution to reproductive study.Structure Biology is a fast developing subject. We analyze the structure of proteins on the foundation of protein purification, crystal screen and optimization, as well as the X-ray diffraction (or other methods). Today more and more researchers are inclined to link function and structure together because they can promote each other.As indicated above,what this paper focus on are about function research of differentially expressed genes and the expression purification,as well as structure analysis of protein,which are introduced as follows:1. In our previous work, RSA-14-44 was identified from rat primary spermatocytes cDNA library constructed by suppressive subtractive hybridization (SSH) between cDNAs,which was trapped by laser capture micro-dissection (LCM) from rat primary spermatocytes and round spermatids.And its GeneID number in GenBank is AY149343. With an open reading frame of 582bp, RSA-14-44 encodes a 193-amino-acid consisted protein. RSA-14-44 was assumed to be a new member of Rho GTPase family because there was a conserved Rho GTPase domain at the N-terminal and it shared high similarity in amino acid sequence with other members of RhoA like subfamily in this domain, and this hypothesis was confirmed by the GTPase activity assay after we got the purified RSA-14-44 protein. At the same time, we purified different mutants of RSA-14-44 and other relative proteins in order to carry through function research experiments.Purified smad3, Jabl and other proteins were also got on the platform of protein purification, which promote their function research efficiently.2.Tte1925 which encodes the hypothetical protein was obtained from Thermoanaerobacter tengcongensis by Zhong Qian (Beijing Institute of Genomics). It was assigned the Genbank accession number NP₆23500. The reading frame encodes a polypeptide consisting of 301 amino acid residues. TTE1925 was assumed to be mutarotase according to the bioinformatic analysis and this hypothesis was approved to be true by the spectropolarimeter. Because of its specialty, tte1925 was subcloned into the prokaryotic expression vector pGEX-6P-1 and overexpression was obtained in the E.coli BL21 (DE3) through transformation of the right recombinant plasmid which has been verified by colony PCR and sequencing. Soluble fusion protein with glutathione S-transferase (GST) tag expressed highly by the induction of isopropylβ-D-thiogalactoside (IPTG) and was purified in a three-step procedure, including Glutathione SepharoseTM 4B affinity, ion chromatography(Resource Q 6mL) and gel filtration chromatography(10/300 superdex200). The purity of purified protein is more than 99% and a large amount of claval crystals whose X-ray diffraction reached 1.9A were obtained, which may contribute to the structure research of TTE1925 and its function illustration.