细胞标签蛋白纯化中的难题及解决方法要点

丝氨酸蛋白酶抑制剂 PMSF，AEBSF-HCl Aprotinin，Chymostatin
Leupeptin
6
快速操作法
Cell lysis
Transfer to tubes
Centrifugation
传统纯化方法
Collect supernantant
Filtration
Protein degradation
10
优化缓冲液条件
蛋白
Histidine tagged Nurr1 ligand binding domain expressed in E. coli
参数研究 结合缓冲液
pH values: pH 6.0 to 8.5 (8 buffers) NaCl: 100 -750 mM Glycerol: 5-10% b-mercaptoethanol: 0.05%
A280
Refolding Gradient: 6 M – 0 M urea 30 ml gradient volume Flow rate: 0.5 ml/min
A280
Column: HisTrap™ FF 1 ml Resin: Ni Sepharose™ FF System: ÄKTAprime™
40.0
ml
mAU
4000
3500
GSTrap HP 1 ml
3000
6.3 mg
2500
2000
1500
1000
500
0 0.0
10.0
20.0
30.0
40.0
ml
Sample:
20 ml unclarified E. coli lysate GST-Hippocalsin
Flow rate: Sample application: 0.3 ml/min,
FT1 E1 FT3 E3 E2 FT2
Run 1. HisTrap HP 1 ml Run 2. StrepTrap HP 1 ml Run 3. HisTrap HP 1 ml + StrepTrap HP 1 ml
Purity
Run 1
Run 3
Run 2
Acknowledgement: Martina Nilsson, Biovitrum Stockholm, Sweden 20
Up to 10 mg 34 µm
mAU 4000
GSTrapTM 4B 1 ml
3000
24.8 mg
2000
1000
0
0.0
10.0
20.0
30.0
40.0
ml
mAU
4000
3500
GSTrap FF 1ml
3000
8.4 mg
2500
2000
1500
1000
500
0 0.0
10.0
20.0
30.0
Elution Gradient: 5 – 500 mM imidazole Flow rate: 1.0 ml/min
Volume
23
标签的酶切和去除
Glutathione GST
Yield (relative units)
1000
800
600
400
200
0
0,1 0,3 0,4
1
1,5
Flow rate (ml/min)
RT Col d
Low yield
13
Low yield
层析填料的选择
Media
Gluthatione
Gluthatione
Sepharose™ 4B Sepharose FF
Gluthatione Sepharose HP
Advantages High Capacity
Higher flow rates High resolution and
possible
concentration
Capacity Bead size
Up to 30 mg 90 µm
Up to 15 mg 90 µm
WastAe3A5A7A9 Waste
80.0
ml
1
2
ቤተ መጻሕፍቲ ባይዱ
Mr
3
4
5
6
7
89
mAU 400
300
200
100 0 20.0
1
3
F
2
A1A2A3A4A5A6A7A8A9 A11A13A15B14B12B10B8B7B6B5B4B3B2B1C1C2C3C4C5C6C7
40.0
60.0
80.0
100.0
ml
97 000 66 000 45 000
2 样品制备
快速操作 低温 加入蛋白酶抑制剂 DNA和RNA酶
3 纯化 快速操作 尽量减少操作步骤
5
避免蛋白降解
金属离子螯合剂 EDTA，EGTA， 塔罗肽（马组链霉菌）
天冬氨酸蛋白酶抑制剂 碘乙酸，PMSF，
Pepstatin，云芝发酵物
蛋白酶
苏氨酸蛋白酶抑制剂 Unknown
巯基蛋白酶抑制剂 PMSF，TLCK， N-乙基顺丁烯二酰亚胺 TPCK，Antipain-HCL
15
纯度检测
Purity
SDS-PAGE
kDa
97 66 45 30 20.1 14.4
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
SuperdexTM 5/150 GL
• 50 µl eluate from StrepTrapTM HP • 10 min
在不同分子量位置有很多条带或峰
Protein purification
Purification of unclarified lysate
Cell lysis
Protein purification
Column: HisTrap™ FF crude Resin: Ni Sepharose™ Fast Flow
更高产量和高生物 学活性蛋白
2. 凝胶过滤
Column: HiLoad™ 16/60 Superdex™ 200 pg Sample: 5.2 ml eluted pool from HisTrap™ System: ÄKTAexplorer ™
mAU 4000 3000 2000 1000
0 0.0
F3
20.0
40.0
F4 60.0
提高纯度
1 多步纯化 2 双标签蛋白 3 优化结合洗脱条件 4 优化螯合的金属离子 5 重折叠和包涵体表达
18
1 两步纯化
Purity
1. 亲和层析
Column: HisTrap™ FF 1 ml
Sample: 50 ml (His)10-Trx-P 450 in E. coli lysate System: ÄKTAexplorer™
Mr
97 000
E
66 000
45 000
30 000 20 100 14 400
1
2
3
4
8
Low yield低产量
Poor binding结合量低 Inefficient elution不充分的洗脱 Media dependence填料的选择
Protein degradation Low yield Purity Tag removal
GST
MBP Strep-tag II
His
Size大小
Media 载量 Capacity
Purity纯度
Increased增溶 solubility
Price价格
26 kDa
>10 mg/ml
40 kDa 10 mg/ml
Medium Medium
8 aa 6 mg/ml
High
6 aa 40 mg/ml
9
低产量
Poor Binding
标签未外露 提取液中的因素影响结合，如生物素 结合缓冲液条件没有优化（IMAC） 配基与标签之间结合动力学很慢
Inefficient Elution
洗脱缓冲液条件没有优化
沉淀（柱上或洗脱后）
Low yield
使用变性剂 添加抗生物素蛋白 优化条件 减小流速
优化条件 减少上样体积 采用线性梯度洗脱 去污剂/NaCl
2. 内源性GST
3. 天然金属结合蛋白
e.g. Cu/Zn Superoxide dismutase 17 kDa 超氧化物歧化酶
4. 富含组氨酸蛋白
e.G Chloramphenicol acetyl transfererase 25 kDa 氯霉素乙酰转移酶
柱上酶切 优化咪唑浓度
17
Purity
转译后修饰产生不均一？
如何检测? Western blot 免疫杂交 Amino acid analysis氨基酸分析
蛋白水解？
共洗脱？
16
共洗脱
anti-DnaK
Purity anti-GST
1. 伴侣蛋白
DnaK -70 kDa GroEL - 60 kDa
在超声前加去垢剂和、或还原剂
Western blot analysis. Samples: Fractions from second IEX step (first step on Glutathione Sepharose™)
50mM Tris pH7.9 0.5mM EDTA 50mM NaCl 5% glycerol
12
动力学：流速的影响
流速对GST结合影响
GST-谷胱甘肽结合动力学较慢
上样阶段流速一定要低
Binding capacity study using GST-(His)6 on Glutathione Sepharose™ 4 Fast Flow
标签蛋白纯化中的难题 及其解决方法
1
纯化中的难题
1 蛋白的降解 2 低收率 3 蛋白纯度 4 去除标签
2
Affinity tags亲和标签
Simplification方便纯化 Increase solubility增加可溶性 Prevent proteolysis防止蛋白水解
Introduction
1
0,00 0
0
10
20
30
40
50
Imidazole conc. (mM)
Imidazole concentration
21
4 优化金属离子
Purity
Metal ions
Histidine tagged proteins
Untagged proteins
Ni Co
+++++ ++++
++
+
Cu
++
++++
Zn
+++ +++++ Strong binding
++ Weak binding
+
Column: HiTrap™ IMAC FF (prepacked with un-charged IMAC Sepharose™ FF)
Cu2+ Zn2+ Co2+ Ni2+
22
Purity
5 柱上复性和his标签蛋白纯化
30 000 20 100 14 400
LMW Start FT wash eluted 1 2 3 F
19
HisTrap™ FF
Superdex™ 200 pg
2 双标签蛋白
Combination of a two-step HisTrap™ HP and StrepTrap™ HP purification Sample: 15 ml Strep II-protein-(His)6 in E. coli lysate System: ÄKTAxpress™
7
未澄清的样品的快速纯化
Protein degradation
Column: HisTrap™ FF crude 1ml Sample: 酵母细胞表达带组氨酸标签的未经澄清的样品15ml Flow rate: 1 ml/min System: ÄKTAexplorer™
E
E
S dil. 2x dil. 4x
Low
FLAG®
8 aa 0.6 mg/ml
Highest
3
蛋白的降解
全蛋白 带标签的降解蛋白
SDS-PAGE
Western blot
Protein degradation Low yield Purity Tag removal
4
避免蛋白降解
1 用蛋白酶缺陷性的表达宿主
BL21-CodonPlus®cells
高通量缓冲液条件筛选
Low yield
His MultiTrap™ FF
Acknowledgement: R. Steele and B. Grasberger,
Johnson & Johnson Pharmaceutical R&D, USA
11
万金油-buffer-TGE
Tris-Glycerol-EDTA
Equilibration, wash & elution: 1 ml/min 14
蛋白纯度
Raising antibodies Crystallization Characterization
> 90-95% > 99% > 99%
Protein degradation Low yield Purity Tag removal
Purity
3 优化咪唑浓度
产量vs纯度
Purity
Abs 490/280 Abs 490
Yield
2,00 1,50 1,00 0,50
Purity Yield
6
5
4 Column: HisTrap™ HP 1ml 3 Sample: GFP-(His)6 expressed in E. coli 2 Detection: 280 and 490 nm