1. Purification of Thermostable DNA polymerase
담당교수 : 이승택
담당조교 : 문선진

2. Purpose Overexpression of Taq polymerase
Purification of Taq polymerase

3. Introduction Taq Polymerase 94 kDa protein from Thermus aquaticus
Thermostable enzyme that replicates DNA at 74°C and exhibits a half-life of 40 minutes at 95°C.
catalyze the polymerization of nucleotides into duplex DNA in the 5´3´ direction in the presence of magnesium.
exhibit 5´3´ exonuclease activity but not 3´ 5´ exonuclease activity.; Taq Pol. Is error-prone.
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)
Extension rates (dNTPs/sec/enzyme molecule) ℃
70 ℃ < 60
55 ℃
37 ℃
22 ℃
5’3’ exonuclease activity: to remove primers from replicon
3’5’ exonulease activity: for proof reading

4. Introduction
- Protein properties and their effects on purification strategies.
Temperature Stablity: Taq.Pol. is stable in high temperature.

5. Strategy of Taq polymerase purification
Introduction
Strategy of Taq polymerase purification
1. Using heat stability heating at 75℃
How is heat stability of Taq polymerase achieved?
- Amino acid substitution
Opposite charge substitution.
Hydrophobic and aromatic substitution.
2. Using DNA binding protein
PEI precipitation.
ion-exchange column chromatography.
DNA
PEI
Taq pol
Taq Pol.은 37C에서 최대 활성을 가지는 일반적인 orgarnism에서 분리한 단백질과는 달리 온천수에서 사는 Thermus Aquaticus에서 분리한 단백질이기 때문에 고열에 안정한 특징을 가지고 있다. Taq DNA polymerase의 thermal stability는 여타 DNA polymerase에 비해 증가된 enzyme core의 hydrophobicity, electrostatic force에 대한 안정성 및 enzyme 표면에 더 많이 존재하는 proline residue들의 존재에 의해 용매 분자들과의 interaction의 향상 등의 여러 원인에 의한 것으로 생각되고 있다.
Bio-rex
resin
(-)

6. Polyethyleneimine (PEI) precipitation
Introduction
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.
PEI has been used as protein precipitant and as a nucleic acid precipitant.
The bulk of the nucleic acids and much of the phospholipids in the extract are removed by precipitation with the synthetic polycation PEI.
PEI is used to precipitate acidic DNA-binding proteins selectively. Thus, a trial extract should be made at a low salt concentration to see whether the protein can be precipitated with PEI and eluted by high salt from PEI precipitant.
In pH 7, PEI is charged positively. In lower pH than 7, the positive charge of PEI is more stable. As the result, positive-charged PEI recruits negative charged molecules (e.g. DNA).

7. Ion-exchange chromatography
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
cation exchangers : (-) charged beads associate with exchange cations.
ex) -SO3- , -COO- , -PO32- , -AsO32-
anion exchangers : (+) charged beads associate with exchange anions.
ex) -NH3+ , =NH2+ , =N+= , =S+-
On a cation exchanger resin, such as Bio-Rex 70 resin, neutral molecules and anions do not interact with the resin.
Usually the resin is used in an ionic form with a lower selectivity for the functional group than the sample ions to be exchanged.
The sample ions are then exchanged when introduced, and can be eluted by introducing an ion with higher affinity for the resin or a high concentration of an ion with equivalent or lower affinity.
Bio-rex 70:
Weak cationic exchangers that provide high-capacity separation of high molecular weight solutes.
This resin contains carboxylic acid exchange groups.

8. Procedure Overview Preparation of Cell lysate Purification
Over-expression of target protein (IPTG induction)
Cell lysis (lysozyme)
Heating at 75℃/1 h (Denaturation of undesired proteins)
Precipitation by PEI
Ion-exchange chromatography
Elution
Preparation of Cell lysate
Purification
Overview
SDS-PAGE
1. Lysozyme cleaves beta (14) linkage between N-acetylglucosamine and N-acetylmuramine in the peptidoglycan of bacterual cell wall.
2. Solublization target proteins by heating. (Denatured, insolublized other proteins is precipitated.)
3. Precipitated Taq pol-DNA-PEI complex.  Elution by high concentration of KCl
4. Ion-exchange chromathgraphy by Biorex-70  Elution by high concentration of KCl

9. PEI preicipitation buffer
Procedure
Buffer preparation
Lysis buffer
PEI preicipitation buffer
Ionic exchage buffers
Buffer A
Buffer B
Buffer C
Reagent
Final conc.
Tris-Cl (pH 7.9)
50 mM
10 mM
HEPES (pH 7.9)
20 mM
Glucose
KCl
0, 25, 50, 150 and 200 mM
EDTA
1 mM
PMSF
5 mM
Tween20
0.5 %
NP40
Tris, Hepes: buffering agent
Glucose: Lysis stabilizer
EDTA, PMSF: Protease Inhibitor
Tween20, NP40: Weak detergent

10. Procedure 1st day 2nd day Seed Culture
; overnight culture a single colony in 3 ml of LA media (LB + ampicillin) at 37 ℃
2nd day
Inoculation of seed culture (0.5 ml) into 50 ml of LA media
Culture cells at 37 ℃ (O.D. 600 nm: ~ 0.5)
Induction of target protein with 0.5 mM of IPTG (by adding 25 ul of 1M IPTG) for 14~16 h at 37 ℃

11. Procedure 3rd Day 1. Harvest :
1) Centrifuge cells at 6,000 rpm for 10 min at 4 ℃
(50 ml centrifugation tube)
2) Discard the supernatant
2. Resuspension:
1) Resuspend cell pellet with 5 ml of Buffer A
(Resuspend중 20 l (~1/500)를 보관한다.  Cell)
# Gel용 시료에는 항상 1/5 vol의 sample buffer를 추가
2) Centrifuge at 6,000 rpm for 10 min at 4 ℃ and discard supernatant
3. Cell lysis :
1) Resuspend cell pellet with 2.5 ml of Buffer A containing
lysozyme (final conc. 4 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 ℃ (Invert sample at every 10 min)
3) Centrifuge the lysate at 12,000 rpm for 10 min at 4 ℃
4) COLLECT supernatant into a fresh tube
(Supernatant 5 ml중 20l (~1/250)를 보관한다.  Lysate Sup.)

12. 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)
2) Incubate sample on ice for 10 min
3) Centrifuge lysate at 12,000 rpm for 10 min at 4 ℃
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 ℃
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
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 5 l (1/600) 보관 = Column input sample )
Role of KCl (1)
25 mM: Washing
150 mM: Elution Taq Pol. from PEI precipitant
Dilution of KCl to 50 mM: To bind to Biorex-70 resin

13. 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 of Buffer C with 50 mM KCl after
resin packing
10. Sample loading :
- Load sample into equilibrated Biorex70 resin column
(Flow through 25 l (1/120) 를 보관한다.  F.T.)
11. Column washing :
- Wash sample-loaded resin with 900 l of Buffer C with 50 mM KCl (5 3ml)
(300 l 씩 두 번 washing, 마지막 300 l washing에서 25 l (~1/12)를 보관한다.
 Washing)
12. Elution :
1) Elute proteins with 0.4 ml of Buffer C with 200 mM KCl
2) Fractionation-collect 0.1 ml of elute per tube
(각 fraction당 2 l (1/50)씩 보관한다.  E1, E2, E3, E4)
3) Add glycerol 25 ul to each eluted sample and store at -70℃.
13. Confirmation of the result: SDS-PAGE
Role of KCl (2)
Dilution of KCl to 50 mM: To bind to Biorex-70 resin
(For 150 mM of KCl, Taq pol. can’t bind to the resin, dilution to 50 mM is necessary.)
Biorex-70 is equilibrated in 50 mM KCl buffer.
50 mM: Taq Pol. bound Biorex-70 resin washing
200 mM: Elution

14. Procedure SDS-PAGE 확인 1) 10 % SDS-PAGE 을 만든다.
   2) 각 Sample에 5× Sample buffer 2 분간 끓인다.
   3) 준비된 각 sample을 SDS-PAG에 loading 한다.
M / Cell/ Lysate Sup./ Column input sample / F.T. / Washing / E1 / E2 / E3 / E4
   4) 100 V 로 dye-front가 gel 끝에 닿을 때까지 전기 영동 한다.
   5) 전기 영동이 끝난 gel을 Coomassie brilliant blue (staining solution)에 넣고 30 분간 staining 한다.
   6) Gel을 destaining solution 에 넣고 1시간 동안 rocking한다.
# 조교가 destaining된 gel 을 scan 하여 생화학과 홈페이지에 게시 예정.
(보고서의 결과에 protein purification yield 및 purification fold (purity)를 추정하여 계산할 것.)

15. Result 1 2 3 Control 2ug 1ug 0.5ug M 1 2 3 4 5 6 7 8 9 10 Marker
2ug 1ug 0.5ug M
(각 step의 전체 부피 중 gel에 loading된 sample의 비율)
<해당 단계의 젤 안에 있는 Taq DNA Pol의 양,
해당 단계의 젤 안에 있는 전체 단백질의 양>
Marker
1 Cell (1/500) < 4 ug / 36 ug>
2. lysate Sup (1/250) < 6 ug / 18 ug>
3. Wash of PEI pellet
4. Column input sample (1/600) < 2.5 ug / 3.5 ug)
5. Flow through (1/120) < 0.5 ug / 0.8 ug >
6. Washing (1/12) < 0.08 ug / ug>
7. Elute 1 (1/50) < 0.25 ug / ug>
8. Elute 2 (1/50) < 1 ug / 1.01 ug>
9. Elute 3 (1/50) < 1 ug / 1.01 ug>
10. Elute 4 (1/50)<0.5 ug / 0.51 ug>
1.Cell
2.Lysate sup
3. Wash of PEI pellet
4.Column input sample
5. F/T
6. wash
7. Elu1
8. Elu2
9. Elu3
10.Elu4
Fig.1. Purification of Taq DNA Polymerase

16. Result
Starting material (1번 cell)을 기준으로 하여 2번 step (heated lysate), 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 (ul)
rate
Target protein in gel (ug)
Target protein in step (ug)
Total protein in gel (ug)
Total protein in step (ug)
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)
Marker 1
Cell 5 10
500 4
2000 36
18000
0.11
　1.000 2
Sup of heated lysate 20
250 6
1500 8
0.75
6.8 3
Column input sample
600
2.5
2.01
1206
1.24
11.27
Flow-through
120
0.5 60
1.51
181.2
Washing
0.3 12
0.08
0.96
0.081
0.97
Elution1
0.1 50
0.25
12.5
0.251
12.6
0.998
9.07
0.006 7
Elution2
1.01 51
0.025
Elution3 9
Elution4 25
0.51 26
0.0125
***
Elution (pool)
138
140.6
0.069