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1 Chap 5 : 구 Interchapter A 단백질의정제 (Protein Purification) 여러단백질중특정단백질을분리하여순도를높이는과정구조적연구, 단백질기능규명, 단백질간상호작용규명, 항체조제등에사용.

2 단백질의정제 (Protein Purification) a) 정제준비 : 대상, 용도, 필요량, 순도, 안정화방법, assay 방법등설정 - 단백질안정화 : glycerol 5~20%, 환원제 (DTT, b-me, TCEP), protease inhibitor(also EDTA) detergents ( 계면활성제 ), 낮은온도 (4 o C) b) 단백질순도측정 : - SDS-PAGE(sodium dodecyl sulfate-poly acrylamide gel electrophoresis) (SDS : CH 3 -(CH 2 ) 10 -CH 2 -O-SO 3 -Na+) AA(CH 2 =CH-CO-NH 2 ) + (APS, TEMED: 촉매 ) ---> polyacrylamide - mass-spec. - HPLC...

단백질의정제 (Protein Purification) 크기에의한분리 a) dialysis ( 반투막 )

gel-filteraton : 다공성 bead ( 큰분자먼저용출 ) 순도및크기확인 (

native-page (-SDS, 자연 ) - 2D-gel (isoelectric

- Isoelectrofocusing ( pi 값 ) cf) staining : commassie vs

3 단백질의정제 (Protein Purification) 크기에의한분리 a) dialysis ( 반투막 ) : 막구경, 삼투압 b) micro-filteration : 막구경 (Mw cut-off) c) gel-filteraton : 다공성 bead ( 큰분자먼저용출 ) 순도및크기확인 ( electrophoresis ( 전기영동 ) : PAGE - SDS-PAGE (+SDS, 변성 ) - native-page (-SDS, 자연 ) - 2D-gel (isoelectric points->molecular weight) --> mass spec. - Isoelectrofocusing ( pi 값 ) cf) staining : commassie vs silver staining cf) blotting : Western (Protein), Southern(DNA), Northern(RNA) 확인 SDS-PAGE staining

4 단백질분리 : low pressure chromatography 방법 Size exclusion (Gel filtration); Affinity chromatography; Ion exchange chromatography; Hydrophobic Interaction chromatography; Purification schemes

5 LC columns Sizing Column :GPC ( Gel-permeation Chromatography) ( 다공성의 beads : 크기에의한분리 ; 큰것먼저용출 ) - molecular shieve, adsorption : cross-linked dextran ( 연한망상구조 -Poly AA) Charged Columns : IC ( Ion-exchange Chromatography) : - Anionic exchange column ( 자체 (+), (-) 하전 -capture) DEAE (Diethyl amino ethyl cellullose ) ; (-CH 2 ) 2 -N+-(CH 2 CH 3 ) 2 - Cationic exchange column ( 자체 (-), (+) 하전 -capture) phosphocellulose(pc) ; -O-PO 3 - ; CMC ( carboxymethyl cellulose) ; CH 3 -COO - Affinity Columns : (Affinity Chromatography) ( 특정결합의특이성을이용하여분리 ) ( 예 ) (PFK)-ATP 칼럼, (hexokinase-glucose 칼럼 ), His-tag column, GST-column Hydrophobic column : 소수성 interaction 이용하여단백질잡아냄. alkyl sepharose 소수성 : C-8 < C-10 < C-12 < C-18 columns phenyl sepharose columns

6 Gel Permeation Chromatography (GPC) : 분자량에의한분리 분자량이서로다른구형단백질의분리에사용된다. 구형단백질의경우분자의크기는분자량 (Mw) 에비례한다. GPC ( 혹은 GFC) 의경우칼럼내에다공성인 bead 를통과하는단백질의이동특성에따라분리한다. 분자량이작은경우는 bead 의모든구멍 (pore) 를통과해나오므로천천히칼럼을통과하게되고, 분자량이큰경우는 bead pore 를통과하지못하므로 ( 크기때문에 ), 더빨리용출된다

7 Ion Exchange chromatography (IC): 단백질하전 (Surface Charge ) 에의한분리 단백질은특정 ph 에서각아미노산들의하전에의해서음혹은양의총하전을띄게된다 ( 단 pi 에서는중성 ). IC 는특정 ph 에서하전을 갖는단백질의특성을이용하여정제한다. 음이온교환칼럼 (anionic exchange column): 자신은양하전 (+), 결합하는단백질은음하전 (-) 을띈다. 예 ) DEAE Cellulose column : O (CH 2 ) 2 -NH + -(CH 2 CH 3 ) 2 양이온교환칼럼 (cationic exchange column) 자신은음하전 ((-), 결합하는단백질은양하전 (+) 을띈다. 예 ) CM Cellulose column : O-CH 2 -COO- phospho cellulose(pc) ;-OPO 3 -

8 Ion chromatography : resin

Affinity chromatography : 단백질의 Biological Activity 나기능에의한분리 단백질 - 기질결합의특이성이용 (kd ~ 10-12 ); 예 ) hexokinase-glucose 결합 glucose (small dark blue molecule) hexokinase (large enzyme).

9 Affinity chromatography : 단백질의 Biological Activity 나기능에의한분리 단백질 - 기질결합의특이성이용 (kd ~ ); 예 ) hexokinase-glucose 결합 glucose (small dark blue molecule) hexokinase (large enzyme). 1) affinity column 제조 : support + spacer + substrate(inhibitor) 2) Specific Binding to column resign, 불순물은 buffer 로닦아냄 3) 결합된효소나단백질의용출 ( 기질사용 ) 4) 결합된기질은투석 (dialysis) 나 gel-filteration 으로제거. 순수한단백질분리

10 Affinity chromatography : his-tag purification Ni 2+

11 CaM cloning with his-tag

Hydrophobic interaction chromatography (HIC): Columns made from matrices having mildly hydrophobic surfaces bind proteins at high ionic strength by interacting with hydrophobic patches on the protein

12 Hydrophobic interaction chromatography (HIC): Columns made from matrices having mildly hydrophobic surfaces bind proteins at high ionic strength by interacting with hydrophobic patches on the protein surface. Generally such columns are loaded in very high salt (e.g. 2M (NH 4 ) 2 SO 4 ), and after loading a reverse salt gradient is run (down to buffer without added salt). In lower ionic strength buffers, the hydrophobic forces holding the protein to the matrix weaken, and the protein elutes at some point in the gradient. Hydrophobic group bound to solid phase Binding Elution high salt (increases water surface tension, decreases available water molecules, increases hydrophobic interactions) decrease salt add detergent decrease polarity of mobile phase

13 UV absorbance UV absorbance UV absorbance UV absorbance 칼럼별분리방법 Larger Protein Free ligand concentration Smaller Protein V 0 V i V t GPC Column Affinity Column Increasing salt concentration decreasing salt concentration Ion exchange Column Hydrophobic Column

14 정제 : 세포에서단백질의분리과정 - homogenization : 세포파괴 * grinding, sonication, freezing/thawing (lysozyme) -differential centrifugation -Salting out (ammonium sulfate precipitation, (NH 4 ) 2 SO 4 ) differential centrifugation

protein-solvent(water) 의안정성이지속되다가 salt 첨가후 salt-solvent 상호작용 ( 이온 -dipole) 으로단백질의물제거로안정화저해 - 단백질

15 정제 : 단백질의선택적침전 - (NH 4 ) 2 SO 4 Fractionation : 단백질의선택적침전 ( 물과이온 -dipole 결합 ), 저온에서잘녹음 a) salting in : 적정선까지첨가시 (solublization) b) salting out : 적정선초과시 ( salt out, 침전 ) (NH 4 ) 2 SO 4 의첨가의경우 : protein-solvent(water) 의안정성이지속되다가 salt 첨가후 salt-solvent 상호작용 ( 이온 -dipole) 으로단백질의물제거로안정화저해 - 단백질 : PI 부분에서불안정, 반경축소, charge balance 상실 -> 침전 salt/ph 에의한단백질용해도변화. b) salt 에의한단백질용해도변화 (salting in/out).

16 순도분석 : Electrophoresis ( 전기영동 ) SDS-PAGE 를이용한순도확인과정 ( 주로단백질에사용 ) ((SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS : CH 3 (CH 2 ) 10 CH 2 OSO 3 -Na+) Agarose gel electrophoresis ( 주로 DNA)

17 Decreasing molecular weight 단백질전기영동 : 단백질순도분석 / 분자량유추 SDS-PAGE 전기영동순서 apply protein to gel well 에넣기전에단백질은변성시킴 (+DTT, +SDS, + heat) AA gel 에도 (+SDS, + DTT) - apply voltage : 100 ~ 200 v ( 약 1 ~ 2 시간 ) : 분자량에의한분리 - staining with dye - 표준단백질의분자량과비교하여미지단백질의순도 / 분자량결정. +

18 NATIVE PAGE : 단백질의 Native 한상태의 Mw 및순도결정 - SDS; -DTT; - 열변성 SDS-PAGE 와는대조적으로 SDS, DTT 와열변성과정등이생략되어서단백질의자연상태의분자량측정에이용된다. 단백질자체가 + 하전을갖는경우는이동하지않아사용할수없다.

19 Native 분자량의결정 : GPC gel permeation chromatography (GPC); native-page, analytical ultracentrifuge GPC 에의한 native Mw 결정 : subunit 결정에도움. 분자량을아는표준물질 ( 단백질 ) 과미지시료를 GPC 에건후 Mw 와 retention time ( 용출부피 ) 의상관그래프에서미지시료의분자량을결정한다. 단점 : 대략적인분자량값만결정된다. 분자량은분자의형태에의존된다. (asymmetric/symmetric)' 정확한분자량은 analytical ultracentrifuge 등에의해결정될수있다. -equilibrium sedimentation - velocity sedimentation -mass-spec (MALDI-TOF)

단백질정제의예 : - UMP synthase : - specific acivity = total activity units / total protein ( 예, 40.4/11,700 = 0.

20 단백질정제의예 : - UMP synthase : - specific acivity = total activity units / total protein ( 예, 40.4/11,700 = ) - unit : total amount of enzyme activity - % recovery = 단계별 total activcity units 100 (%) 초기 activity total unit

21 단백질정제의다른예 Specific activity : total activity /total protein Percent recovery : (activity/initial activity) * 100%

22 단백질의 1 차구조 ( 순서 ) 결정 단백질성분결정단백질순서결정

23 단백질의성분 (amino acid composition) 결정 - 고순도의단백질준비 ( 순수할수록좋음, 적어도 >95%, ) - Amino Acid Composition 결정 (6 M HCl, 100~110oC 36 hrs acid hydrolysis->hplc) polypeptide(protein) --> AAs( 펩타이드결합절단 ) polypeptide 가각각의 amino acid 로잘라져서나옴 * 주의사항 : 3 개아미노산 Asn(N) ---> Asp (D) : N 과 D 는 N+D D 로나옴 Gln(Q) ---> Glu (E) : Q 와 E 는 Q+E E 로나옴 Trp (W) --> destroyed : spectroscopically 결정 (Trp, 흡광상수 )

24 단백질의 1 차구조 ( 순서 ) 결정 : N- 말단결정 N- and C-terminal 결정 - N-terminal 결정 : N- 말단의유도체화 -fluorodinitrobenzene - dansyl chloride Dansyl 에의한 N- 말단유도화반응 : N- 말단확인

25 - C-terminal 결정 ) - carboxypeptidase 효소처리 (C- 말단에서부터순차적절단 : 시간조절중요 ) 단백질의 1 차구조 ( 순서 ) 결정 : C- 말단결정

26 단백질의 1 차구조 ( 순서 ) 결정 : 효소에의한절단 -digestion (protease or chemicals) : 작은펩타이드로나눔 ( 짧은펩타이드가 Edman degradation 반응에서신뢰도가높으므로!) 효소사용절단 : 특정한아미노산순서인식및절단 - 효소절단부위 pepsin F,W or Y 전 trypsin K or R 후 chymotrypsin F, Y or W 후 papain K or L, R &G 후

27 단백질의 1 차구조 ( 순서 ) 결정 : 화합물에의한절단 chemical 사용절단 - BrCN : M 다음절단 peptide + BrCN--> N-homoserine lactone + C-terminal + CH 3 SCN H 3 C H 2 C N H S CH 2 CH C O H N R' O + H 3 C S H 2 C Br displacement of Br CH 2 C CH N N C H O H N C R' N O C-terminal Peptide + H 2 C - BNPS-skatole ; C-terminal W H 2 C O N H C C O H Homoserine lactone N H H 2 C C H H 2 C C O N H 2 O Methyl thiocyanate H 3 C S C N + - NH 2 OH ; N-G bond ( 아스파라긴 - 글라이신결합절단 ) - 분리 : separation (HPLC or TLC...) - 절편된각펩타이드들의순서결정 - 전체순서결정 (disulfide bond 위치포함 ) : overlapping 되는펩타이드간의상관관계에의해분석

28 단백질의 1 차구조 ( 순서 ) 결정연습 Correct sequence: L-V-G-K-A-E-F-S-G-I-T-P-K Trypsin cleavage: A-E-F-S-G-I-T-P-K L-V-G-K Chymotrypsin Cleavage: L-V-G-K-A-E-F S-G-I-T-P-K Edman degradation: L

29 단백질의 1 차구조 ( 순서 ) 결정연습 :insulin B chain 의순서 예

30 Edman degradation : 짧게잘린펩타이드의순서결정 N-terminal residue+ phenylisothiocyanate --> phenylthiocarbamoyl-peptide (1) (1) + HCl (or trifluoroacetic acid (F 3 CCOOH)) --> phenyllthiohydantoin 유도체 + (n-1)-peptide (N-말단쪽절단 ) (Edman degradation)

31 N-&C- Terminal Protein Sequencer Hewlett Packard(USA)

32 단백질순서결정의중요성 - 동종단백질간의일차순서비교 : 중요 domain 발견, 단백질기능유추 ( 하나단백질전체순서는주로 DNA 순서에서결정된다 ) - cloning 을위한정보제공 (primer design) : AAs--> DNA primer--> PCR 에의한 full-sequence DNA clone fishing - 짧은펩타이드의경우구조결정의보완자료로사용가능 NMR (solution) or X-Ray (solid)

33 단백질의 1 차구조 ( 순서 ) 결정연습 Amino Acid Analysis yielded: Asn, Gly, Leu, Lys, Met, Tyr, Trp Trypsin had no effect; Edman degradation yielded PTH-Tyr; CNBr treatment yielded a tetrapeptide of positive charge and a tripeptide of zero charge at ph 7.0; Brief chymotrypsin (cleaved at Trp) yielded a dipeptide and a pentapeptide which contains Gly, Leu,Asn, Lys, and Met; NH 2 OH 처리 : dipeptide (G, K) + pentapeptide ( 나머지 ) What is the sequence of the peptide?

34 연습문제 ( 계속 ) 다음에답하시오. A mixture of three proteins (A, B, and C, see below) can be theoretically separated by ion exchange chromatography using cellulose coated with diethylaminoethyl (DEAE) groups followed by a gel filtration column. In the first step, the mixture was applied to the resin at ph 8.0 and washed with the same buffer to elute proteins that did not bind. The resin was then washed with a buffer ph 6.0 in order to elute the protein that bound to the column. Protein A is a homodimer with a molecular weight = 50,000 Daltons and a pi=5.00 Protein B is a homotetramer with a molecular weight =160,000 Daltons and pi = 7.0 Protein C is a monomer with a molecular weight = 75,000 Daltons and pi = 9.0 Questions: A). When one analyzes the mixture of the three proteins on a SDS-PAGE & native PAGE gels, where would each of the protein stop on the those two gels (draw schematic gel pictures and label each band with name of the protein)? B). Which protein(s) is initially bound to the DEAE column? C). Which protein(s) is eluted from DEAE at ph 6.0? D).The DEAE column is further washed with a NaCl gradient. Is there more protein eluted and which one? E). Eluted proteins from both steps C) and D) are combined and subjected to a gel filtration procedure. Draw the elution profile of the gel filtration procedure and label each peak with their names.

35 다음펩타이드순서를보고답하시오. A-E-K-F-V-C-Y-M-G-F 1) Trypsin digestion의산물? 2) +Succinic anydride + trypsin? 3) +ethyleneimine + trypsin? 4) +Chymotrypsin? 5) +CNBr? Hint : Lys (K) + succinic anhydride trypsin resistant Cys(C) + ethyleneimine lysine like (trypsin subtrate)

7 lb/in 2 ) (peptide : 주로 reverse phase column 사용 ) - LPC (Low Pressure Chromatography) ; 1~2 atm (open column) - FPLC (Fast

36 단백질의정제 (Protein Purification) Liquid Chromatography - HPLC (High-Performance Liquid Chromatography : 약 5 atm 이상적용가능정지상과이동상의분배, 흡수력차 : 용출순서차이. 주로유기용매사용, 펩타이드등의분리에적합 ( 단백질은변성 - 유기용매,. pressure ;100 psi = 100 lb/in 2 (1atm = 760Torr = 14.7 lb/in 2 ) (peptide : 주로 reverse phase column 사용 ) - LPC (Low Pressure Chromatography) ; 1~2 atm (open column) - FPLC (Fast Performance Liquid Chromatography, 자연 ) : less pressure than HPLC ex) AKTA purifier (Pharmacia) 정성분석 : LC-MS 예 ) Shimadzu LC/MS 2010 ( 분리 - 질량분석 ( ~ 2000))

37 단백질특성분석기기 Circular Dichroism (CD) : 2 차구조 MALDI-TOF : 질량분석 (150 ~ 1,000,000 Da or up)

38 단백질특성분석기기 : 4 차구조, 분자량 Analytical Ultracentrifuge ( 초원심분석기 ) 분취용 : 시료분리 분석용 (XL-A) : 분광기기 ( 중합상태분석 ) 최소 10 rpm to 최대 60,000 rpm ( 약 250,000 x g).

39 목포대학교공동실습관생체고분자분석 Facility 시료정제 LC, FPLC Primitive analysis Advanced analysis 고분해능구조분석장비 400MHz NMR XRD 질량분석 MALDI-TOF 중합상태판정 XL-A 이차구조판정 CD 분석의뢰 자연상태상호결합연구 XL-A 고분해능형태분석장비 SEM 저분해능구조 - 성능분석형광기

Microsoft PowerPoint - Chapter 3-2

Microsoft PowerPoint - Chapter 3-2 Chapter 3 단백질의탐구및단백체학 (3) 겔거르기 (Gel filtration) chromatography: 크기에의한분리 - Resin: 다공성구슬 = Sephadex, Sepharose, Biogel = Bead size: 100 mm - 원리 : 분배 (partition) chromatograph = 분배 : 용질이이동상 (mobile phase)