Study Of Saccharomyces Cerevisiae Cdc48Protein On Expression In Prokaryotes,purification And Preliminary Electron Microscope Structure

Saccharomyces cerevisiae can be a kind of important model organism to study theeukaryotes. Yeast genes and genes of many diseases have a high homology. Structural studies ofthese proteins, helps to deepen the understanding of these diseases, so that the level of diagnosisand treatment of these diseases can be improved.Cdc48is a member of type Ⅱ AAA ATPase family. Cdc48in the mammal homologknown as p97or VCP. Biochemical characteristics of Cdc48include ATPase activity, ubiquitinUbiquitin binding and with association with diverse cofactors. Cdc48play a role in many cellularevents and associated with many human malignant diseases.Through the structure research homolog p97of Cdc48demonstrated that the protomer ofCdc48consists of two Walker-type ATPase domains, D1and D2, which are flanked by anamino-terminal N domain and a carboxy-terminal, unstructured tail. Cdc48consists of sixmonomer to form a complex, around a central axial hole with a Zn2+.Study on the structure of p97is part of the analysis of some parts of the domain, the otherpart is obtained structure lacks the N terminal and C terminal part of the amino acid.Study onCdc48protein mainly focuses on its function, not much research on the structure of it,It sets alimit to research the function of Cdc48.We imported the plasmid contained Saccharomyces cerevisiae full-length cdc48gene intoE.coli and expressed the protein. We optimized the conditions of expression successfullyexpressed soluble Cdc48protein at a temperature of16with adding0.05mM IPTG overnight.We optimized conditions of nickel ion affinity purification. We use the1M NaCl in thebuffer.We added20mM iminazole and0.6%Triton X-100in the lysis buffer to reduce thenonspecific miscellaneous proteins binding the bead.In order to improve the binding of theCdc48protein and bead, we purified the protein after incubation with the bead. In the cleaningprocess, first of all, we used a lot of cleaning buffer A containing50mM imidazole and0.6% Triton X-100. secondly, we used a small amount of cleaning buffer B containing100mMimidazole to clean the bead. To ensure protein in less volume was eluted, we used elute buffercontaining500mM imidazole in the elution process.We obtained high purity Cdc48protein through the nickel ion chelating beads affinitypurification, DEAE anion exchange chromatography and Superose6gel filtrationchromatography.By electron microscopy, with fewer samples and dye fast observed the structure feature ofCdc48image, acquired low resolution Cdc48six protein dimer ring model. This identified thequality of Cdc48protein is good and protein status is relatively uniform.My subject is not only the first step to analysis of the study for the frozen three-dimensionalelectron microscopy for high resolution, but also the key step.It has important significance tocomprehensive analysis the fine3D structure of Cdc48protein, establish its structural changesunder different conditions and the interpret the biological mechanism of function.