2013 年度修士論文

2013 年度修士論文
ヤツメウナギの祖先型ヘモグロビン Node 25 タンパク質の
合成および昨日解析
指導教授
常重アントニオ
指導博士研究員
松尾高稔
大学院工学研究科
生命機能学専攻修士課程
学生証番号 11R7113
マクシンイチ
膜晋一
1.
1
2.
2
3.
3
4.
4
4.1
(pET-Node25)
BL21(DE3)
4
6
7
8
4.2 Node25
4.3 pET-Node25
4.4
Node25
4.4.1
Node25
8
4.4.2 QSepharose
8
4.4.3
9
CMSepharose
4.4.4
Node25
4.5
4.6
SDS-PAGE
Node25
Node25
CD
4.6.1 CD
12
13
13
4.6.2 Oxy
Node25
4.6.3 Deoxy
CD
Node25
4.6.4
14
CD
15
Node25
15
4.7
Node25
4.7.1
4.7.2
10
16
16
Node25
17
5.
18
5.1
5.2
5.3 Node25
(pET-Node25)
Node25
18
18
19
5.3.1 Q Sepharose F.F.
Node25
5.3.2 CM Sepharose
Node25
1
19
20
5.3.3 Lowry
5.4
5.5
5.6
21
Node25
Node25
Node25
CD
Hill plot, P 50
nmax
5.6.1
22
23
24
pH
5.6.2
Node25
Hill Plot, P50
Node25
nmax
Hill Plot, P50
pH7.4
5.6.3
24
nmax
27
Node25
nmax pH7.4
Hill Plot, P 50
30
32
33
33
34
2
(Hb)
2
Hb
oxy
deoxy
Hb
Hb
Hb
Hb
ML
tree
Hb
Node25
pGEM- Node25
Node25
Node25
SDS
CD
14 kDa
CD
Node25
HbA
pH
Hill
60 µM
Node25
pH
Bohr
Node25
HbA
Node25
60 600
Hb
1
1,200
2.1
(
)
2
1
6
1
6
43
(Hb)
)
41
6
(
(
1)
(
)
)
Hb
2.2
5
2)
3
(Hb)
Hb
2
(Mb)
Hb
Hb
)
(Oxy)
(Deoxy)
(
)
Hb
Hb
Hb
(
Mb
Mb
Hb
Hb
Hb
2,3)
4,5)
2.3
DNA
2
2.4
Hb
(NCBI)
Hb
15
65
5
ML-tree
node23
Node23
node25
Node25
150
node23
Hb
453
node25
Node23
6, 7, 8)
Node23
Hb
CD
CD
HbNode23
Node23
HbA
(pH8.4)
(pH6.4)
Hill
Bohr
1
pH
1.59~1.36
pH
ATP 2.3-DPG
Node23
2.5
Hb
(OEC)
(pO2)
Hb
pO2 Clark
Hb
Y
X-Y
Hb
(560 nm)
OEC
(Deoxy)
U-4000 (
)
,Pr + O2
)
O2
Y = [PrO2]/([Pr] + [PrO2])
Hb
(Oxy)
PrO2 (Pr
Deoxy
Hb
3
Deoxy
Deoxy
Hb
Hb
560 nm
Clark
0.8 V
(Ag)
(Au)
: O2 + 4H+ + 4e: 4Ag + 4Cl
-
: 4Ag + O2 + 4H+ + 4Cl
2O
O2
Hb
(pO2)
50 %
P50 [mmHg]
(log[Y/(1-Y)] vs. log pO2)
Hill
nmax
Hb
Hb
Hb
Node25
Hb node25)
Node25
4.1
(pET-Node25)
BL21(DE3)
Hb
(ML-tree)
node25
Node25
Node25
pGEM-Node25
<
>
pGEM- Node25
(pET22b(+)
4
Node25
10x H buffer
Ligation Mix
TaKaRa Ex Taq
10x Ex Taq
dNTP Mixture
XL
-
LB
SOC
4% X-gal
IPTG(100 mg/ml)
(50 mg/ml)
BL21(DE3)
<
(BigDynamics Laboratory)
>
pET-22b(+) pGEM-Node25
37
1 µl
2 µl
2 µl
10x H Buffer
3 µl
DDW
22 µl
Total
30 µl
1.0%
(AGE)
DNA
DNA
16
25
DNA
1
1 µl
pET-22b(+)
4 µl
Ligation Mix
5 µl
Total
10 µl
XL1 Blue
100 µl
5
5 µl
20
42
60
SOC
1 ml
5
37
1
LB+Amp
PCR
Node25 F-primer
- TTCGATTCATATGCCGATTGTGGATAGCGGC -
Node25 R-primer
- AAGAATTCAATCACTAGTGAATTCGCGGCCG PCR
TaKaRa Ex Taq
0.1 µl<PCR>
10x Ex Taq Buffer
95
1 min
95
30 sec
55
15 sec
72
1min
1 µl
dNTP Mixture
0.8 µl
10 µM Node25 F-primer
0.5 µl
10 µM Node25 R-primer
0.5 µl
x30
1 µl
DDW
6.1 µl
Total
10 µl
4
PCR
1.5%AGE
pET-Node25
LB+Amp
SDS
4.2
pET-Node25
Node25
<
>
4.1
(pET-Node25/BL21(DE3))
100 mg/ml
(
)
1 M IPTG
(
)
TCA
(
)
<
>
4.1
(pET-Node25/BL21(DE3))
160 rpm
O.D. 600
30
0.716
ml
6
1 M IPTG
1 mM
ml
IPTG
0
10
50
TCA
ml
100%
1200 rpm
SDS-PAGE
4.4.4
4
SDS-PAGE
Node25
4.3 pET-Node25
<
30
1,3,5
100%TCA
4
160 rpm
9)
>
pET-Node25
100 mg/ml
(
)
LB
1
(
BD)
0.5
(
BD)
0.5
NaCl
(
)
TB
1.2
(
BD)
2.4
(
BD)
0.4
(
17 mM
(K HPO )
72 mM
(KH PO )
)
(
)
(
(100 mg/ml)
1 MIPTG
)
(
)
(
)
(Alfa Aesar)
28
<
(
>
pET-Node25
BL21(DE3)
(100 mg/ml)
TB
)
2 µl
LB
2 ml
100 ml(
ml
)
180 rpm
TB
30
150 rpm 37
2
1
6
900 ml(
)
3
2
180 rpm 30
32
1 M IPTG
7
1ml
2
MilliQ
13 ml)
IPTG
15
(28
3 ml
15 ml
15 mg
15
5 6
GRX-220(
7,000 rpm
4
5
)
50 ml
MX-301 (
)
7,000 rpm 4
5 min
-80
4.4
4.4.1
<
Node25
node25
10)
>
4.3
Q Buffer(pH 7.4) 20 mM Tris-HCl
1 M Tris-HCl (pH8.0)
60 ml
(
11.5 N HCl
(
11.5 N HCl
<
)
pH 7.4
)
Milli-Q
0.5 M MgCl
(
)
0.1 M MnCl
(
)
DNase I(10 mg/ml)
(
)
>
4
2
Q Buffer
1
30
1/50
30
0.5 M MgCl
DNase I
CO
4.4.2
1
1/100
0.1 M MnCl
1 ml
30
4
Q Sepharose
<
>
Q Sepharose F.F.
(GE
1 M Tris
10
)
(
(PEI)
(
8
)
)
CO
(
YM10(
(
<
63.5 mm)
20)
)
(
)
(
)
>
CO
4
GRX-220 (
)
13,500 rpm
45 min
1 M Tris
(
pH 8.0
10
PEI
0.3 ) 10
GRX-220
12,500 rpm 4
15 min
YM10
)
10 20 ml
(16 h
)4
8200(
Q Buffer
CO
10
Q Buffer
Q Sepharose
Q Buffer
Node25
Q Sepharose
200 drop/tube
FC 203B
(Gilson)
SDS-PAGE
SDS-PAGE
YM10
)
10 mM
8200 (
Buffer (pH5.8)
(16
(GE
)
)4
4.4.3 CM Sepharose
<
>
CM Sepharose
10 mM
Buffer (pH5.8)
(
Y M 10 (
(
<
63.5 mm)
20)
)
(
)
(
)
>
10 mM
Buffer (pH5.8)
10 mM
Node25
9
CM Sepharose
Buffer(pH5.8)
100 drop/tube
FC 203B
(Gilson)
SDS-PAGE
SDS-PAGE
Node25
YM10
8200(
50 mM
)
Buffer (pH7)
Node25
Buffer
CO
4.4.4
4
Node25
<
SDS-PAGE11, 12)
>
30
29.2 g
N ,N -
(
0.8 g
Milli-Q
)
(
)
(
)
100 ml
(APS)
N,N,N ,N -
(TEMED)
(
)
1.5 M Tris-HCl (pH 8.8)
(
)
Tris
18.2 g
(
)
SDS
0.4 g
Buffer
(
)
2 ml
Milli-Q
(
)
100 ml
(CBB) R250
Milli-Q
1g
(FlukaChemika)
225 ml
(
)
50 ml
(
)
100 ml
(
)
300 ml
(
)
225 ml
Milli-Q
700 ml
Precision Plus
Dual Color Standard
Buffer
10
(
)
0.5 M TrisHCl (pH6.8)
Tris
6.1 g
SDS
0.4 g
Milii-Q
Buffer
(
)
4.2 ml
(
)
0.8 g
(
)
5 ml
2-
8 ml
(
)
4 ml
(
)
40 ml
10 mg
Milli-Q
25 mMTris
)
Buffer
5g
(
0.192 M
SDS
Milli-Q
<
(
20 ml
SDS-PAGE
0.1%
)
100 ml
SDS
Milli-Q
(
)
14.4 g
(
)
1g
(
)
1
>
SDS-PAGE
Laemmli
11, 12)
15
30%
SDS
SDS
Buffer
APS
15
Milli-Q
5
TEMED 10
SDS-PAGE
SDS-PAGE
10
20 mA
15
70~90
SDS
30
9 ml
Buffer
4.5 ml
11
Buffer
SDS-PAGE
Milli-Q
4.5 ml
APS
TEMED
10
Total
18 ml
Total
5
l
2
l
13
l
20
l
SDS-PAGE
4.5
5
Node25
50 mM
Buffer (pH 7.0)
5 ml
13, 14)
Glucose 6-phosphate
(
)
Glucose 6-phosphate dehydrogenase
(
)
NADP
(
)
(
)
(
)
Ferredoxin NADP reductase
20 µl
Ferredoxin
Catalase
(
SOD(super oxide dimutase)
(
N
CO
(
CO
)
)
Node25
)
(
)
(
)
(
)
U-2800
CO
HbA
Node25
13.4
50 mM
Buffer (pH7.0)
12
50
50
Node25
(4
(16h
)
)
Node25
650~450 nm
Node25
CO
Node25
7
20
13, 14)
2
2 mm
50
Node25
N
3
40
(Deoxy
(Deoxy)
)
Node25
Node25
650~250 nm
20
(Oxy)
Deoxy
20
3
(CO)
Node25
Node25
Oxy
10 mm
<
Node25
Oxy
Deoxy
4.6
4.6.1
Node25
650~250 nm
CO
CO
(Oxy)
Node25
Node25
CD
CD
>
Node25
13, 14)
Glucose 6-phosphate
(
)
Glucose 6-phosphate dehydrogenase
(
)
13
NADP
(
)
Ferredoxin NADP reductase
(
)
Ferredoxin
(
)
Catalase
(
)
SOD (Super Oxide Dimutase)
<
(
)
>
45 M Node25
(pH7)
(pH7)
U-2800
45 M
Node25
5 ml
mM
Milli-Q
Buffer(pH7)2.5 ml
Milli-Q
2.5 ml
100 mM
(pH7)
)
(pH7)
<
Buffer(pH7)2.5 ml
(pH7)
Oxy
4.6.2
100
5 ml
45 M Node25
(4
2.5 ml
Oxy
45 M Node25
(pH7)
7
13, 14)
Node25
20 l
2
CD
>
45 M Node25
(pH7)
(pH7)
30
2 mm
(pH7) 800 l
U-2800
650~250 nm
J-820
10
480~245 nm
10
Oxy
45 M Node25
(pH7)500 l
U-2800
2 mm
650~250 nm
Oxy
J-820
5
480~245 nm
U-2800
Oxy
14
3
4.6.3
<
Deoxy
20
Node25
CD
>
Oxy
Node25
(pH7)
2 mm
300 l
800 l
Node25
(pH7)
U-2800
Deoxy
Deoxy
4.6.2
Node25
Node25
10
480~245 nm
Deoxy
20
4.6.4
<
Node25
15)
>
Oxy
Node25
(pH7)
1 M NaOH
<
>
Oxy
M NaOH
Node25
(pH7)1 ml
0.4 ml, 1
0.6 ml
U-3010
600~500 nm
Milli-Q
2 ml
10 mm
Node25
10 mm
Node25
4
590 nm
15
600~500 nm
4.7
4.7.1
Node25
<
>
Node25
1.4 ml
2.1 ml
0.1 M Bis-Tris +0.2 M HCl Buffer (pH 6.4, 6.9, 7.4)
3.5 ml
0.1 M Tris + 0.2 M HCl Buffer (pH7.9, 8.4)
3.5 ml
13, 14)
Glucose 6-phosphate
(
)
Glucose 6-phosphate dehydrogenase
(
)
NADP
(
)
(
)
(
)
Ferredoxin NADP reductase
20 µl
Ferredoxin
Catalase
(
)
SOD (Super Oxide Dimutase)
(
)
50 mM ATP
(
)
(
)
0.1 M Bis-Tris + 0.2 M HCl Buffer (pH7.4)
50 mM DPG
0.1 M Bis-Tris + 0.2 M HCl Buffer (pH7.4)
50 mM IHP
(
)
0.1 M Bis-Tris + 0.2 M HCl Buffer (pH7.4)
<
>
Node25
1.4 ml
2.1 ml
0.1 M Bis-Tris +0.2 M HCl Buffer (pH 6.4, 6.9, 7.4)
Buffer (pH7.9, 8.4)
Buffer
20
3.5 ml
0.1 M Tris + 0.2 M HCl
7
2
30
Oxy
pH
Node25
CO
Node25
650~450 nm
(pH7.4)
50 mM
16
280 l Node25
1.4 ml Milli-Q
Buffer(pH7.4) 3.5 ml
3
1.82 ml
7ml
0.1 M Bis-Tris + 0.2 M HCl
3
(pH7.4)
(pH7)
20 l
7, 8)
2
Node25
(1.2 mM, 0.6 mM)
Node25
1.2 mM
7 ml
50 mMBis-Tris + 0.1 M HCl Buffer (pH7.4)
4
16
1.2 mM
mM)
7 ml
7 ml
(1.2
7
7, 8)
40 l
(pH7.4)
0.6 mM
1.2 mM
(pH7.4)
3.5 ml
2
(pH7.4)
0.1 M Bis-Tris+0.2 M HCl Buffer (pH7.4)
7ml
4.7.2
Node25
<
>
4.7.1
60 uM Node25
7 ml
13, 14, 15, 16)
N
Oxy
Node25
Deoxy
560 nm
Deoxy
Deoxy
Oxy
Deoxy Deoxy
X-Y
17
Oxy
Oxy
5.
5.1
(pET-Node25)
4.1
Node25
pGEM-Node25
pET-22b(+)
pET-Node25
pET-Node25
Node25
pET-22b(+)
LB
>
,
,
20
PCR
4.1
AGE
4.2
1
5.2
Node25
Node25
Node25
4.2
SDS-PAGE
1
14 kDa
14kD
Node25
SDS-PAGE
18
<
IPTG
IPTG
1~5
0
14 kDa
Node25
IPTG
(0
)
TCA
5.3 Node25
5.3.1 Q Sepharose F.F.
Node25
Node25
DNaseI
DNA
Node25
(
)
(
CO
PEI
(
)
)
CO
Q
Sepharose F.F.
SDS-PAGE
15
SDS-PAGE
2
4.4.4
(M) 10
No.3
25
10
(No.3
14 kDa
2 Q Sepharose
SDS-PAGE
19
25)
Lane M
2
14 kDa
SDS-PAGE
Node25
No.3~25
14 kDa
Node25
No.9~17
10, 22, 30 kDa
No.3~25
28 ml
(
10 mM
5.3.2
8200
)
Buffer (pH5.8)
CM Sepharose
(4
)
Node25
CM Sepharose F.F.
SDS-PAGE
3
2
4.4.4
SDS-PAGE
(M) 10
No.1
8
10
(No.1 8)
kDa
14 kDa
3 CM Sepharose
SDS-PAGE
20
No.1 8
14 kDa
SDS-PAGE
Node25
5.3.1
No.1 8
YM10
(
8200
)
50 mM
Buffer (pH7.0)
(4
Node25
SDS-PAGE
10
)
Buffer
9 ml
(
CBB
)
4
Node25
(kDa)
20 ml
SDS-PAGE
5
M
15 kDa
14 kDa
10 kDa
4
Node25
SDS-PAGE
4
Q Sepharose
2
CM Sepharose
Node25
21
5. 3. 3
Lowry
5.3.1
16,17)
5.3.2
)
Node25
(
Node25
Lowry
1. Node25
(ml)
(mg/ml)
(mg)
120
10.77
53.88
120
10.65
53.28
50
14.01
70.08
28
4.54
9.08
20
5.59
11.08
9
13.99
27.98
5.4
Node25
Node25
20
Fe
Node25
4.7.1
7
4.6
CO
(pH7)
50 µM
Oxy
2
13, 14)
Node25
(4
)
Node25
(Oxy Deoxy CO)
(
N
5)
Deoxy
CO
22
Node25
2 mm
Node25
(pH7)
Oxy
5 Oxy
Deoxy
Deoxy
Oxy
CO
CO
410
430 nm
Node25
(pH7)
Node25
Soret
530
580 nm
Q
Node25
Node25
Hb
Node25
Node25
5.5
Node25
6(A)
Node25
CD
6(B)
Oxy
CD
Hb
Deoxy
420 nm
CD
Soret
18, 19)
Hb
CD
Hb
Node25
23
(A)
(B)
J. Lampe
:
282-284 (1972)
6 : Node25 A
5.6
5.6.1
Hb
Node25
Node25
pH
4.7.1
pH
B
CD
Hill Plot, P50
Hill Plot, P50
nmax
60
Node25
nmax
7 ml
Oxy
N
Oxy
Deoxy
Oxy
24
Deoxy
Oxy
Deoxy
560 nm
pH
Node25
Hill Plot
60
HbA
(pH7.4)
nmax
Hill plot
8
P50
Hb
3
pH
7
Hill Plot, P50
Node25
nmax
3
2.5
2
1.5
1
0.5
0
0
0.5
1
1.5
2
2.5
3
ph6.4
pH6.9
-0.5
pH7.4
-1
pH7.9
pH8.4
-1.5
HbA pH7.4
-2
pH
pO2
Node25
mmHg
25
2
pH
Node25
P50
pH
P50 (mmHg)
nmax
6.4
27.1
1.49
6.9
25.9
1.52
7.4
23.8
1.61
7.9
21.2
1.65
8.4
20.1
1.64
9.5
2.26
HbA 7.4
8
pH
Hb
26
nmax
Hill plot
3
pH
pH
Hb
Node25
P50
nmax
HbA
pH8.4
pH6.4
Bohr
Hill
1.64 (pH8.4)
1.49 (pH6.4), 1.52 (pH6.9), 1.61 (pH7.4), 1.65 (pH7.9),
pH
Hill
60
pH
5. 6. 2
nmax pH7.4
Node25
Node25
Hill Plot, P50
Node25
60
Node25
(pH7.4)
2 mM
(2,3-DPG ATP IHP)
(pH7.4)
(pH7.4)
Hill plot
Node25
P50
nmax
Hb
10
9
Hill plot, P50
5
27
nmax
2
1.5
1
0.5
0
0
0.5
1
1.5
2
2.5
3
-0.5
node25
pH7.4+ATP
node25
pH7.4+IHP
node25 pH7.4
-1
node25
pH7.4+DPG
-1.5
9
Node25
4
Node25
Hill plot
P50
Effector
P50 (mmHg)
nmax
None
23.8
1.61
2 mM IHP
26.1
1.51
2 mM ATP
24.6
1.56
2 mM DPG
25.5
1.55
28
nmax
10
Hb
5
Hb
ATP IHP
DPG
Hill plot
P50
Node25
5
Hb
Hill
Hill
29
1.5
nmax
5. 6. 3
nmax
Node25
pH7.4
node25
Hill plot
Hill Plot, P50
(60
600
1,200
11
)(pH7.4)
Node25
P50
Hb
plot, P50
nmax
Hill
12
3
2.5
2
1.5
1
0.5
0
0
1
2
3
-0.5
HbA 60uM
-1
1.2 mM
600 uM
-1.5
60 uM
-2
12
Node25
7
Node25
(µM)
P50
P50 (mmHg)
nmax
HbA
60
9.5
2.26
Node25
60
23.8
1.61
Node25
600
29.8
1.69
Node25
1200
33.3
1.74
30
nmax
nmax
12
Hb
7
Hb
Node25
60
(pH7.4)
HbA
600 1,200
31
Hill plot
P50
nmax
Node25
12
Hill
Hb
Node25
1.61, 1.69, 1.74
Node25
Hb
HbNode25
pET-Node25
IPTG
Node25
Node25
pI
6.73
CM Sepharose
(pH5.8)
Node25
Node25
Node25
Hb
pI
Node23
pI
pI
6.73
8.61
Node25
Node25
Node25
Hb
Node25
Hb
pH
HbA
Bohr
pH
Hb
ATP
IHP
Node25
Node25
Hb
Hb
Node25
(ML-tree)
3
Hb
5
Node23
Hb
ATP IHP
32
DPG
Node25
ML-tree
Node25
Hb
3
Node23
Hb
pH
ML-tree
Hb
Hb
Mb
Node25
Hb
Hb
CD
Node25
Node25
Hb
pH
Bohr
Hill
nmax
pH
Node25
Node25
Node25
Hb
Hb
1.
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