Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Homology modeling wikipedia , lookup
Circular dichroism wikipedia , lookup
Protein mass spectrometry wikipedia , lookup
Intrinsically disordered proteins wikipedia , lookup
Western blot wikipedia , lookup
Protein structure prediction wikipedia , lookup
Protein–protein interaction wikipedia , lookup
Protein purification wikipedia , lookup
List of types of proteins wikipedia , lookup
Nuclear magnetic resonance spectroscopy of proteins wikipedia , lookup
研習報告與討論 • Crystallization of the Lysozyme • The Structure of HDV Antigen 姓名:強伍翎 學號:u891602 研習地點:中研院分生所 N209 Summer Research(2002/7~8) ~ in Institute of Molecular Biology Academic Sinica R.O.C[1] Dr. Chwan-Deng (David) Hsiao[2], 強伍翎(u891602) Crystallization of the Lysozyme The Structure of HDV Antigen • Crystallization of the Lysozyme Abstract hydrolyzes the β-1,4 glucosidic linkages between N-acetylmuramic acid and N-acetylglucosamine in the cell wall of certain microorganisms.[6]We used the protocol from other lab and successfully got the crystals. Background Crystallization:formation of solid crystals from a homogeneous solution. It is essentially a solid-liquid separation technique. Crystals are grown in many shapes depending upon the conditions they crystallized. Their shapes include cubic, tetragonal, orthorhombic, hexagonal, monoclinic, triclinic, and trigonal. Process: (1) nucleation--the growth of a new crystal. To initiate the process, supersaturation driving force is necessary. (2) growing--crystals grow gradually by surface interaction with the solute. But how do we reach supersaturation and let crystal seeds grow slowly? Vapor diffusion method is a common approach: A drop composed of the mixture of sample and precipitating agent is placed in vapor equilibration with a liquid reservoir of reagent. Typically it contains a lower reagent concentration than the reservoir. To achieve equilibrium, water vapor leaves the drop and eventually ends up in the reservoir. As water leaves the drop, the sample undergoes an increase in relative supersaturation. Both the sample and reagent increase in concentration as water leaves the drop for the reservoir. Equilibration is reached when the reagent concentration in the drop is approximately the same as that in the reservoir. (1)sitting drop (2)hanging drop Material and Method Solution A: 6% lys(0.1M CH3COONa) Solution B: 15% NaCl(0.1M CH3COONa) Sitting Drop: Every well 5λA+5λB, reservoir B 1ml, 25℃ Hanging Drop: Every well 1λA+1λB (in cover slips), 500λB, 25℃ Result The next day, I found that in all wells there were lots of large clear crystals with cubic, tetragonal, hexagonal, and trigonal shapes. In addition, the crystals in the sitting drop are bigger than that in the hanging drop. • The Structure of HDV Antigen Abstract HDV (hepatitis D virus)[7] is a satellite virus of hepatitis B virus. The encoded delta antigen is a nuclear phosphoprotein with RNA binding activities in regions near N terminus (residue 24~50). The crystal structure from residue 12 to 60 has been solved, and now we try to find how this region binds with polynucleotides. We now have about 11 conditions that needed modifying to gain proper crystals for X-ray diffraction. BackgroundThe tequniques that we used in the experiment: Ion-exchange chromatography: The charged resins interact differently with various proteins, thus separate them by charge. X-ray diffraction: The atomic planes of a crystal cause an incident beam of X-rays to interfere with one another as they leave the crystal and are detected and calculated to get the structure. Material and Method Buffer I: 50mM hepes, 20% glycerol, pH 7.8 Buffer II:50mM hepes, 2M NaCl, 20% glycerol, pH 7.8 PET plasmids with Lac operon and AmpR E.coli (competent cell for transformation) Insert DNA corresponds to residue 14 to 59 of HDAg[3] DNA segments from 9 to 23 nucleotides[4] Transformation 2μl DNA+100μl competent cell on ice 5 min 42℃ 2 min on ice 30 min Cell culture +1ml LB 37℃ shacking 2 hr Transfer to 1L LB with 1ml amp 37℃ shacking 12 hr Get the proteins Collect cell pellet by centrifuge at 4℃, 4Krpm 20min Resuspend pellet in 15ml buffer I Microfuidizer to break the cells Centrifuge at 4℃, 25Krpm 40min Run PCcolumn Wash with 0.5, 0.8M NaCl buffer(add buffer II to I) stepwise Elute with 1.2M NaCl buffer Concentration Transfer eluted solution to Amicon concentrator 5kd cut until the concentration larger than 10μg/μl (OD595nm using BSA as standard protein) Dialysis Just dilute the salt NaCl Set screen Add protein solution to DNA fragment (1:1) Add 5M NaCl (1:2) Modify conditions X-ray diffraction Result I set screen I、II、III、V [5]and found that microcrystals grew under about 11 conditions: 4.6 Na Acetate 3%MPD 0.02M CaCl2 Snow dust 0.4M (NH4)2HPO4 Needle 6.5 Na Citrate 30% PEG4K 0.2M NH4OAc 8.5 Tris 50% MPD 0.2M (NH4)2PO4 5.5 Mes 5% PEG8K 200mM KCl 7.5 Tris 10% MPD 10mM MgCl2 7 Hepes 5% PEG4K 200mM NH4OAc 150mM Mg(OAc)2 Needle 7 Hepes 10% PEG400 100mM KCl 10mM CaCl2 Column 7 Hepes 10% PEG400 100mMKCl 10mM MgCl2 Needle 6.5 10% Cacodylate Hexanediol 5mM MgCl2 0.1mM Co(NH3)6Cl3 Needle 100mM MgSO4 200mM KCl Needle 5.5 Mes 10% PEG400 Rectangle Aggregated needle 10mM MgCl2 Rectangle Needle Discussion From the table above, we can see that there are no obvious common features between these conditions. Maybe the most important is that the protein has the supersaturated condition which drives the protein to aggregate orderly to become crystals. Future Work Microcrystals are not big enough for x-ray diffraction. The conditions I got are still needed further modifying. Besides, the N-terminal delta antigen used in the experiments are linked to His tag. Maybe cleaving this fusion protein to get pure portion of the protein that we actually want to solve is another way the lab can try to have the crystals. Reference 1 中研院分生所 2 Dr. Chwan-Deng (David) Hsiao 蕭傳鐙博士 Crystallographic Studies of Various Biological Macromolecules 1982 B.S. Dept. Chemistry, Chung-Yuan Christian Univ. 1984 M.S. Dept. Chemistry, Natl. Taiwan univ. 1993 Ph.D. Dept. Crystallography, Univ.of Pittsburgh, USA 1993-95 PDF IMB, Academia Sinica 2/95-5/99 Assistant Research Fellow, IMB 5/99-present Associate Research Fellow, IMB Recent Research: Hsc 70, HDV antigen , Phosphoglucose isomerase and Toc 34 3 Amino acid sequence of HDAg from 14 to 59: MSRSERRKDRGGREDILEQWVSGRKKLEELERDLRKVKKKIKKLEEDN PWLGNIKGIIGKKDKDGEGAPPAKKLRMDQMEIDAGPRKRPLRGGFTDKERQ DHRRRKALENKRKQLSSGGKSLSREEEEELKRLTEEDEKRERRIAGPSVGGVN PLEGGSRGAPGGGFVPSMQGVPESPFARTGEGLDIRGSQGFPWDILFPADPPFS PQSCRPQ 4 5 6 7 DNA segments from 9 to 23 nucleotides: 5’ CTA GTG GGA ACG TCG TCG TCG CT 3’; decrease by two nucleotides from both ends each time There are ten screens that are got commercially.