Download Dr. Chwan-Deng (David) Hsiao[2], 強伍翎(u891602)

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.