The Study On Protein Expression, Purification And Solution Structure

The research on gene express regulation becomes an important aspect in the field of life science, in which post-transcription is an even hotter issue. The mRNA degradation plays an important role in post-transcriptional regulation of gene expression by controlling the number of translation times. The mRNA degradation in E. coli involves two steps. The first step is a rate-determining process, which converts the mRNA 5'terminus from a triphosphate to a monophosphate. In the next step, the endonuclease cleaves the modified substrates in the first step. Previous studies indicate that members of the Nudix hydrolase family have been identified in various organisms and shown to possess the mononucleotide pyrophosphohydrolase activity, the first step of mRNA degradation. Recently, it was reported that RppH(previously named as NudH)is a pyrophosphohydrolase, a member of the Nudix family, which removes pyrophosphate from the RNA 5'ends. Multiple sequence alignment indicates that the N-terminal residues among the members of Nudix family are well conserved, which is essential for the pyrophosphohydrolase activity. However, the molecular mechanism of the biological function of RppH(RNA pyrophosphohydrolase) remains unclear thus far.Currently, the X-ray, EM and NMR are the main ways in the study of protein structure. Compared with X-ray and EM, NMR possesses irreplaceable advantages. Firstly, protein often makes its function in solution and NMR can determine its solution structure. This structure can truly set out the structure of protein in normally physiological state. Secondly, NMR can provide dynamic information of protein in various time scales, which is important to its physiological functions. It gives much information on the relationship between molecule structure and biological function. However, there are also some disadvantages in NMR, such as high cost, long cycle-time, not valid for large protein (>30 KDa).RppH is cloned and expressed in E. coli by gene engineering. The RppH protein was purified by cation-exchange chromatography and FPLC. After find the proper conditions of protein expression and purification, we express the protein with 13C and ¹⁵N labeled. We determine the solution structure of RppH protein from E. coli by NMR (BMRB ID: 16124, PDB ID:2kdv). It reveals that RppH consists of fiveα-helices and eightβ-strands, forming a sandwich structure. Electrostatic surface of the RppH model shows that there is a channel in the structure. The positively charges assemble on the dorsum surface of the channel. The three-dimension RppH structure shows significant similarities to other protein structures of Nudix family in E. coli though they have low amino acid sequence identities. And then, we investigate the molecular mechanism of RppH biological activity. Previous study shows that Mg2+ acts as a co-factor of the proteins of Nudix family. The chemical shifts of amide hydrogen and nitrogen of RppH do not change after the addition of Mg2+. The ATP titration experiments, which is a substrate analog of RppH, indicate that the chemical shifts of Rpph change after the addition of ATP. The residues, which chemical shifts changed significantly, assemble near the channel of the structure. Previous study shows that there is also a channel in the structure of the Yeast homologue protein Dcp2, which is predicted as a RNA binding channel.