Purification of
Thermostable DNA polymerase
담당교수 : 이승택 교수님
담당조교 : 남윤성, 김동현
Purpose
1. Overexpression of Taq polymerase
2. Purification of Taq polymerase
(Analyzing the purification steps and result)
Introduction
Taq Polymerase
1) 94 kDa protein from Thermus aquaticus
2) Thermostable enzyme that replicates DNA at 74°C
and exhibits a half-life of 40 min. at 95°C
cf) DNA polymerase I from mesophile E. coli  had to insert every PCR cycle
http://www.turbosquid.com/3d-models/dnapolymerase-protein-3d-max/630742
Extension rates of Taq polymerase (dNTPs/sec/enzyme molecule)
75 oC
55 oC
37 oC
22 oC
150
24
1.25
0.25
3) catalyze the polymerization of nucleotides into duplex DNA in the 5´3´
direction in the presence of Mg2+
* Optimal reaction condition: 1.4 mM MgCl2, 50~55 mM KCl, pH 7.8~9 (8.3) at room temp (corresponding to pH7.2 at 74oC)
4) exhibit 5´3´ exonuclease activity but not 3´ 5´ exonuclease activity
; It is ‘Error-prone’
Introduction
- Protein properties and their effects on purification strategies.
Sample and target protein properties Influence on purification strategy
Temperate stability
Need to work rapidly at lowered temperature
pH stability
Selection of buffers for extraction and purification
Selection of conditions for ion exchange, affinity or reversed phase
chromatography.
Organic solvents stability
Selection of conditions for reversed phase chromatography
Detergent requirement
Consider effects on chromatographic steps and the need for
detergent removal. Consider choice of detergent.
Salt (ionic strength)
Selection of conditions for precipitation techniques, ion exchange
and hydrophobic interaction chromatography
Co-factors for stability or activity
Selection of additives, pH, salts, buffers
Protease sensitivity
Need for fast removal of protease or addition of inhibitors
Sensitivity to metal inos
Need to add EDTA or EGTA to buffers
Redox sensitivity
Need to add reducing agents
Molecular weight
Selection of gel filtration media
Charge properties
Selection of ion for affinity medium
Biospecific affinity
Selection of ligand for affinity medium
Post translational modifications
Selection of group-specific affinity medium
Hydrophobicity
Selection of medium for hydrophobic interaction chromatography
Introduction
Strategy of Taq polymerase purification
1. Using heat stability  heating at 75℃
How is heat stability of Taq polymerase achieved?
Although the tertiary structure of both DNA polymerases are simlar,
Comparing with DNA pol. I in E. coli, Taq polymerase has more ionic bonds,
hydrophobic interactions, and decreasing flexibility of peptide bonds
by Amino acid substitutions (LysArg / GlyAla).
+++++++
2. Using that Taq pol. is ‘DNA binding protein’
------DNA
 PEI precipitation.
------+++++++
3. Using ion-exchange column chromatography.
(-)
+
+
PEI
++
-- - Taq pol
++
(-)
Bio-rex
resin
(-)
Introduction
Strategy of Taq polymerase purification
Polyethyleneimine (PEI) precipitation
- Monomer (Ethyleneimine): It consists of a three-membered ring.
The corners of molecule consist of -CH2- linkages and =NH.
- Polyethyleneimine: A converted form into a highly branched polymer with about 25 % primary amine
groups, 50 % secondary amine groups, and 25 % tertiary amine groups.
- Function: neutralization of excess anionic charge (in other words, PEI recruits negative charge molecules),
especially under acidic and neutral pH condition.
Polyethylenimine
+
Introduction
Ion-exchange chromatography
Ion exchange :
an exchange of ions between two electrolytes.
used to denote processes of purification, separation of
aquous and other ion-containing solutions.
Type of ion-exchanger
http://www.waters.com/waters/ko_KR/HPLC-SeparationModes/nav.htm?cid=10049076&locale=ko_KR
cation exchangers : (-) charged beads associate with exchange cations.
ex) -SO3- , -COO- , -PO32- , -AsO32anion exchangers : (+) charged beads associate with exchange anions.
ex) -NH3+ , =NH2+ , =N+= , =S+-
Bio-rex 70:
Weak cationic exchangers that provide high-capacity separation of high molecular weight solutes.
This resin contains carboxylic acid exchange groups.
Procedure
Overview
Over-expression of Taq pol.
(IPTG induction)
Preparation of
Cell lysate
Cell wall breakdown by
lysozyme (cell lysis)
Heating at 75℃/1 h
(Denaturation of undesired proteins)
Purification
Precipitation of
Taq pol.-DNA complex by PEI
Elution of Taq pol. (high salt)
Ion-exchange chromatography
Elution of Taq pol. (high salt)
SDS-PAGE
Procedure
Buffer preparation
Buffer A (for lysis):
- 50 mM Tris-HCl (pH7.9) : pH buffering
- 50 mM Glucose : Osmotic stabilization
- 1 mM EDTA : Protease inhibitor
- 0.5 % NP40 : decreasing non-specific binding, protecting protein-protein aggregation
Buffer B (for precipitation):
- 10 mM Tris-HCl (pH7.9)
- 50 mM KCl: providing protein structure, keeping DNA-Taq. Poymerase bidning complex
- 1 mM EDTA
- 1 mM PMSF: protease inhibitor
- 0.5% tween 20: decreasing non-specific binding, protecting protein-protein aggregation
- 0.5% NP40
Buffer C (elution for ion exchange chromatography):
- 20 mM HEPES (pH7.9): pH buffering, buffer for ion exchange chromatography
- 0, 25, 50,150, 200 mM KCl
- 1 mM EDTA
- 5 mM PMSF
- 0.5% tween 20
- 0.5% NP40
Procedure
Buffer preparation
For lysis
For PEI precipitation
For eluting ionic exchange
Buffer A
Buffer B
Buffer C
Reagent
Final conc.
Reagent
Reagent
50 mM
Glucose
50 mM
KCl
50 mM
KCl
0, 25, 50, 150
and 200 mM
EDTA
1 mM
EDTA
1 mM
EDTA
1 mM
PMSF
1 mM
PMSF
5 mM
Tween20
0.5 %
Tween20
0.5 %
NP40
0.5 %
NP40
0.5 %
0.5 %
10 mM
HEPES
(pH 7.9)
Final conc.
Tris-Cl
(pH 7.9)
NP40
Tris-Cl
(pH 7.9)
Final conc.
20 mM
Procedure
1st day
1. Seed Culture
overnight culture a single colony in 3 ml of LA media (LB +
ampicillin) at 37 oC
2nd day
1. Inoculation of seed culture (0.5 ml) into 50 ml of LA media
2. Culture cells at 37 oC (O.D. 600 nm: ~ 0.5)
3. Induction of target protein with 0.5 mM of IPTG (by adding
25 ul of 1M IPTG) for 14~16 h at 37 oC
Procedure
3rd Day
1. Harvest :
1) Centrifuge cells at 6,000 rpm for 10 min at 4 oC
(50 ml centrifugation tube)
2) Discard the supernatant
2. Resuspension:
1) Resuspend cell pellet with 5 ml of Buffer A
(Resuspend 시료 중 16 l (~1/300)를 보관한다.  Cell)
# 전기영동 할 시료에는 항상 1/4 vol의 5x sample buffer를 추가
2) Centrifuge at 6,000 rpm for 10 min at 4 oC and discard supernatant
3. Cell lysis :
1) Resuspend cell pellet with 2.5 ml of Buffer A containing lysozyme (final conc. 4.5 mg/ml)
2) Incubate it for 15 min at RT (room temperature) with Rotator
4. Denaturation of undesired proteins:
1) Add 2.5 ml of Buffer B to cell lysate
2) Incubate cell lysate for 1 h at 75 oC (Mix sample by inverting at every 10 min)
3) Centrifuge the lysate at 12,000 rpm for 10 min at 4 oC
4) COLLECT supernatant into a fresh tube
(Supernatant 5 ml중 16 l (~1/300)를 보관한다.  Heat-stable sample)
Procedure
3rd Day- continued
5. Precipitation :
1) Add neutralized 10% polyethyleneimine(PEI) to sample slowly
(drop by drop) up to 0.15% final conc. (75 ul of 10% PEI)
Precipitation by PEI
2) Mix well and incubate sample on ice for 10 min
3) Centrifuge lysate at 12,000 rpm for 10 min at 4 oC
4) Discard supernatant
6. Washing :
1) Resuspend protein pellet with 1 ml of Buffer C 25 (25 mM KCl)
2) Centrifuge sample at 12,000 rpm for 10 min at 4 oC
3) discard sup.
7. Elution by resuspending pellet with 1 ml of Buffer C 150 (150 mM KCl)
1) Several strokes in a Dounce homogenizer
2) Centrifuge sample at 12,000 rpm for 10 min at 4 oC
3) Collect the supernatant!!
8. Dilute supernatant with 2 ml of Buffer C (0 M KCl) up to 50 mM final conc.
 Now, sample is ready to be loaded!!
(PEI ppt에서 Elution한 시료 10 l (1/300) 보관 = Column input sample )
Procedure
3rd Day- continued
9. Resin packing & equilibration :
1) Load 0.1 ml of Biorex70 resin into disposable column
2) Equilibrate resin with 10 bed volume (1 ml) of Buffer C with 50 mM KCl after resin packing
10. Sample loading :
- Load sample into equilibrated Biorex70 resin column (6 drops/min)
(Flow through 10 l (1/300) 를 보관한다.  F.T.)
11. Column washing :
- Wash sample-loaded resin with 900 l of Buffer C with 50 mM KCl
(300 l 씩 두 번 washing, 마지막 300 l washing에서 15 l (1/20)를 보관한다.
 Washing)
12. Elution :
1) Elute proteins with 0.8 ml of Buffer C with 200 mM KCl
2) Fractionation-collect 0.2 ml of elute per tube
(각 fraction당 10 l (1/20)씩 보관한다.  E1, E2, E3, E4)
13. Analysis of aliquots taken during steps : SDS-PAGE
Procedure
SDS-PAGE 확인
1) 10 % SDS-PAGE 을 만든다.
2) 각 Sample을 2 분간 끓인다.
3) 준비된 각 sample을 SDS-PAG에 loading 한다.
M / Cell/ Heat-stable sample/ Column input sample / F.T. / Washing / E1 / E2 / E3 / E4
* cell 시료는 DNA를 분쇄하기 위하여 sonication을 해야 하므로 조교가 미리 준비해 놓은 sample
을 전기영동에 사용하도록 한다.
4) stacking gel= 100 V / running gel= 150 V 로 dye-front가 gel 끝에 닿을 때까지
전기 영동 한다.
5) 전기 영동이 끝난 gel을 Coomassie brilliant blue (CBB) solution에 넣고 30 분간
staining 한다.
6) Gel을 destaining solution 에 넣고 1시간 동안 destaining한다.
보고서 작성 시
1. Handwriting
2. 친구 보고서를 베끼거나 서로 상의해서 적지마세요 (둘 다 0 점 처리).
3. 조교가 destaining된 gel 을 scan 하여 생화학과 홈페이지에 게시하면,
gel 사진과 함께 보고서의 결과에 Homework 1)번과 2)번에 있는 protein purification
yield 및 purification fold (purity) 계산한 표를 작성하세요.
보고서의 고찰에 Homework 3)번과 5)번 질문의 답을 작성하세요.
4. 보고서는 s304호 앞에 상자에 넣어주세요.
Deadline: pm 12:00 (1반- 11일(화) / 2반-12일(수) / 3반- 13일(목) )
늦게 제출시 감점
Result
M
1
2
3
4
5
6
7
8
9
200
116
97.4
66
1. Cell (1/300)
2. Heat-stable sample (1/300)
3. Column input sample (1/300)
4. Flow through (1/300)
5. Washing 3 (1/20)
6. Elution 1 (1/20)
7. Elution 2 (1/20)
8. Elution 3 (1/20)
9. Elution 4 (1/20)
45
31
10 % SDS-PAGE gel
Result
Starting material (1번 cell)을 기준으로 하여 2번 step (heat stable sample), 3번 step (column input
sample) 및 최종 elution (Bio-Rex 70 ion exchange) 후 얻은 단백질(E1-E4)의 정제 step별 purification
fold 및 yield의 계산
Lane
Sample
Total
volume
of step
(ml)
Gel
loading
volume
(㎕)
Ratio of
loading
volume
out of
total
volume
Target
protein
in gel
(㎍)
Target
protein
in step
(㎍)
Total
protein
in gel
(㎍)
Total
protein
in step
(㎍)
Purity of
each step
(Target
protein /
Total
protein)
Purification
fold (Purity
of each step
/ Purity of
the starting
material)
Yield (Target
protein in each
step / Target
protein in the
starting material)
1
Cell
5
16
300
1.0
300
20
6000
0.05
1
1
2
Heat stable
sample
5
16
300
0.5
150
2.5
750
0.22
4.4
0.5
3
Column input
sample
3
10
300
0.4
120
0.55
165
0.72
14.4
0.4
4
Flow-through
3
10
300
0.25
75
0.35
105
5
Washing 3
0.3
15
20
0.125
2.5
0.125
2.5
6
Elution1
0.2
10
20
0.1
2
0.11
2.2
7
Elution2
0.2
10
20
0.7
14
0.71
14.2
8
Elution3
0.2
10
20
0.2
4
0.21
4.2
9
Elution4
0.2
10
20
0.1
2
0.11
2.2
0.96
19.2
0.073
***
Elution (pool)
22
22.8
참고: 일반적으로 step 별 yield가 80% 수준에 도달하는 것이 바람직함.
Homework
1) 각 step 별로 purification yield (수득율 : amount of the target protein in each
step/ amount of the target protein in the starting material) 를 계산하시오. 단,
elution 한 sample들은 pool 하였을 때의 시료로 계산할 것.
2) 각 step 별로 purification fold (정제비 : purity of the target protein in each
step/purity of the target protein in the starting material)를 계산하시오. 단,
elution 한 sample들을 pool 하였을 때의 시료로 계산할 것.
3) Taq polymerase 정제 과정에서 해당 단백질의 손실이 가장 컸던 정제 step을 답하고
원인을 분석하시오.
4) Taq polymerase 정제 과정에서 가장 필수적이라고 생각하는 정제 step을 답하고 그
이유를 답하시오.
5) 실험에 사용 된 buffer A, B, C 에서 각 chemicals의 역할을 간략히 답하시오.