Techniquese in Biotechnology

Sc K
Tng quan v Sc k Phn loi Phng php sc k trao i ion Nhn xt

Tng quan v sc k
Khi nim chung
Sc k l tn gi chung ca mt nhm phng php tch cc cht. Tn gi ny sau c gi nguyn mc d nhiu cht phn tch khng c mu.

Tng quan v sc k
Lch s
Nm 1903, nh khoa hc ngi Nga Svet ln u tin s dng ct hp ph (than hot tnh) tch cc sc t thc vt t mt hn hp ban u (thnh cc lp c mu khc nhau), t t tn cho phng php l sc k (chromatography -ghi mu, theo gc t Hylp chromato- mu sc cng ng thi bao hm ngha tn ca ng trong ting Nga). Tn gi ny sau c gi nguyn mc d nhiu cht phn tch khng c mu.

Tng quan v sc k
Phng php ny sau mt thi gian b lng qun li c pht trin mnh t sau 1930, vi cc pht kin v. Sc k trao i ion (1935), Sc k trn giy (1940), Sc k kh (1940- 50), Sc k lp mng (1938- 50), Sc k lc gel (1959).

Tng quan v sc k

Nguyn tc chung
Nguyn tc chung ca mi phng php sc k l da trn s phn b cc cht gia 2 pha:mtc nh v mt di ng.

Phn

loi

C th phn chia sc k thnh 3 nhm da trn hin tng ho l xy ra trong qu trnh tin hnh:
Sc k hp ph. Sc k phn b Sc k trao i ion (tio).

Mt cch phn loi khc da trn hnh dng cht gi ca pha c nh hoc bn cht ca pha di ng:
Sc k trn ct Sc k trn giy Sc k lp mng Sc k kh.

Cc phng php sc k lng tch protein

Sc k trao i ion
(Ion- exchange chromatography - IEC)

Gii thiu chung

Tng tc ion - c s ca vic tch v tinh ch protein bng IEC- c ng dng thnh cng t cui nhng nm 1940.
Theo nhng thng k mi nht, IEC l k thut tch protein c s dng nhiu nht trong cc quy trnh tinh ch (c th bao gm nhiu bc v nhiu k thut khc nhau):
IEC: 75%, sc k i lc (AC): 60%, sc k lc gel (GF): 50% cc trng hp.

Nhng s liu thng k t cc cng b gn y (t 7 tp ch ho sinh hc uy tn nht th gii trong 2 nm 1996- 1997) cho thy IEC hin c vn l phng php ph bin nht trong cc bc sc k ca quy trnh tinh sch protein (vi c protein thng v protein ti t hp): chim khong 40% (AC: 16,5% , GF: 17%).

Sc k trao i ion
(Ion- exchange chromatography - IEC)

Nguyn tc ca IEC:
Trc tin, protein s gn thun nghch vi cc cht trao i bng tng tc ion gia cc nhm mang in tch tri du, Sau , protein gn c chit rt ring bit, thng l nh vic tng dn lc ion (khin cho tng tc ion b b gy hon ton) Hay dng nht l gradient ca NaCl) ca m (c rt nhiu loi khc nhau c s dng cn bng cht trao i), C th bng cch thay i pH (t dng hn) khin cc nhm tng tc trn protein b mt in tch Mc d y l phng php thng dng trong nhiu phng th nghim ho sinh, c ch c bn trong tng tc gia protein v b mt tnh in vn cn t c hiu bit.

Sc k trao i ion
(Ion- exchange chromatography - IEC)

The principle of ion-exchange chromatography (salt gradient elution)

Sc k trao i ion
(Ion- exchange chromatography - IEC)

The principle of ion-exchange chromatography

The fractionation of variously charged molecules (squares) on an anion exchanger (cirles)

Sc k trao i ion
(Ion- exchange chromatography - IEC)
IEC illustration The separation of an amino acid mixture at pH 3.0 on EIC column (above); Automatically recorded highperformance liquid chromatographic analysis of amino acids on a cationexchange resin (below)

Sc k trao i ion
(Ion- exchange chromatography - IEC)

Nhng nguyn nhn gii thch s thng dng ca IEC

C phn gii cao, Kh nng lin kt protein ln,

a dng (cc loi nha trao i khc nhau, nhiu cch kt hp gia m v pH),
Nguyn tc tch trc tip (cn c trn khc bit in tch), D dng tin hnh. Tuy nhin, v protein l nhng cht cao phn t, c ch tch ca chng thng l phc tp, ph thuc vo nhiu yu t: Khc bit trong s phn b in tch trn b mt phn t,

Sc k trao i ion
(Ion- exchange chromatography - IEC)
C s ca IEC l s cnh tranh cc nhm tch in tri du trn cht trao i gia cc ion ca cht cn tch vi cc ion khc.

Tng tc gia cc phn t nh v cht trao i ph thuc vo in tch thc v vo lc ion ca mi trng.
Khi nng ion cnh tranh thp, cc ion cn tch s lin kt vi cht trao i, Khi nng ion cnh tranh cao, cc ion cn tch s ri ra. Tng tc gia protein v cht trao i cn ph thuc vo: S phn b in tch b mt ca protein. ph. Bn cht cc ion trong dung dch. Cc cht c thm vo nh dung mi hu c. c tnh ca cht trao i.

Sc k trao i ion
(Ion- exchange chromatography - IEC)
R rng l in tch ca protein cng cao, n cng gn cht vo cht trao i tch in tri du. Tng t nh vy, cht trao i cng tch in nhiu (c ngha l c mc thay th cc nhm tch in mnh hn), s lin kt vi protein mnh hn cc cht tch in yu. Nhng iu kin khc nh pH lm thay i hoc protein hoc cht trao i s nh hng n tng tc ca chng v c th c s dng tc ng ln qu trnh trao i ion.

Sc k trao i ion
(Ion- exchange chromatography - IEC)

Nguyn tc ca IEC l phn tch da trn s khc bit v ion

Gm c pha tnh (ct nhi gel, vi khc bit l c cc nhm chc nng dng ion ho) Pha ng.

V cch thc hin IEC gm hai bc: 1) gn protein vo cc in tch c nh trn ct 2) chit rt chng ra.

Sc k trao i ion
(Ion- exchange chromatography - IEC)

Cc cht (nha) trao i ion v cch la chn

Nhiu loi nha c cha nhm tch in c nh trn cht gi khng tan c bn v s dng rng ri (lit k trn bng vi khong pH thch hp). Cc cht trao i c cha hoc nhm acid hoc base: Cht trao i ion base c cha nhm tch in dng v c gi l anionit (nha trao i cation, v chng gn v trao i cation),

Ngc li, cht trao i acid c cha nhm tch in m l cationit (nha trao i anion, v chng gn v trao i anion) (Cc nha tch in dng c gi l "trao i anion"; cc

Sc k trao i ion
(Ion- exchange chromatography - IEC) Cc nha pha rn thng dng gm: cellulose, dextran, agarose, v polystyrene.

Nhm trao i anion yu: DEAE (diethylaminoethyl) l 1 base yu (c in tch dng r khi ion ho v do vy s lin kt vi cc anion trao i), pH 2-9; cht gi c th l dextran agarose, cellulose dng ht hay si
Nhm trao i cation yu: CM (carboxymethyl): l 1 axit yu c in tch m r khi ion ho, do vy lin kt vi cation trao i; pH 3- 10, cht gi c th l dextran agarose, cellulose dng si

Sc k trao i ion
(Ion- exchange chromatography - IEC)
Protein l nhng cht lng tnh, c tng in tch thay i tu thuc mi trng: Dng pH thp, m pH cao Zero pI . Nhm tham gia tng tc ca protein thng l carboxyl: - COOH - COO- + H+ v amino hoc amin bc ba: - NH2 + H+ -NH2H+

Do vy mt cht trao i anion lin kt vi protein thng qua nhm carboxyl khng proton ho v c tch (ri) ra bng nhm amin proton ho i vi cht trao i cation th ngc li.

Sc k trao i ion
(Ion- exchange chromatography - IEC)

Theo nguyn tc chung:

Cc protein khc nhau v lng gc Asp v Glu (a.a axit) tng s s c tch bng nha trao i anion Cc protein khc nhau v Lys, Arg v His s c tch bng nha trao i cation. Tuy nhin cn nhiu tc nhn khc nh hng n s tch. mnh ca lin kt lin quan n c pI ca protein ln s in tch tng s. Do vy, hai protein c pI bng nhau khng tch c bng ng in cn bng li c th tch bng IEC nu s dng gi tr pH m ti chng c cha lng gc tch in khc nhau ng k trn 1 phn t.

Sc k trao i ion
(Ion- exchange chromatography - IEC)

C th la chn kiu tch protein bng in di trn gel polyacrylamid trong iu kin acid v kim, nhng phn tch ny s l nhng kiu phn tch sc k trao i cation v anion tng ng. V d nu tch phn tch tt mt hn hp bng in di kim th n c th tch tt bng cht trao i anon pH tng t. Cellulose l loi nha c a dng hn c tch protein , Nhng dn xut ca Sephadex G-50 b thay i th tch rt mnh khi lc ion thay i Cc dng nha mi ca cellulose, vi ht v ht ca Whatman c lp li, kh nng v phn gii cao hn dng si c. Dung tch vi protein l tng i ging nhau cc loi nha: (0.110.15 g albumin/ ml ca cc loi dn xut DEAE

Sc k trao i ion
(Ion- exchange chromatography - IEC)

Schematic morphological structure of different support materials

Sc k trao i ion
(Ion- exchange chromatography - IEC)

Cch chun b v ti sinh ct

Cn ra, lm trng (mt s nha c bn dng c trng sn c th dng ngay), v x l nha thnh dng cn thit trc khi dng. Cc IEC c th ti s dng nhiu ln, sau mi ln s dng nht thit phi ti sinh (hi nguyn) nha.
Ra v lm trng trn nha vi khong 10 th tch m, lng, gn b cc ht l lng( m bo tc chy n nh).

Charged groups
Functional groups used on ion exchangers
Anion exchangers Functional group
-O-CH2-CH2-N+H(CH2CH3)2 -O-CH2-CH2-N+H(CH2H5)2-CH2-CHOH-CH3 -O-CH2-CHOH-CH2-O-CH2-CHOH-CH2-N+(CH3)3

Diethylaminoethyl (DEAE) Quaternary aminoethyl (QAE) Quaternary ammonium (Q)

Cation exchangers Carboxymethyl (CM) Sulphopropyl (SP) Methyl sulphonate(S)

Functional group
-O-CH2-COO-O-CH2-CHOH-CH2-O-CH2-CH2-CH2SO3-O-CH2-CHOH-CH2-O-CH2-CHOH-CH2SO3-

Sc k trao i ion
(Ion- exchange chromatography - IEC)

Sephadex ion exchangers (SIE)

(A-25 or A-50 and C-25 or C-50 designating the matrix porosity)
Types DEAE Sephadex QAE Sephadex CM Sephadex SP Sephadex A-25 A-50 A-25 A-50 C-25 C-50 C-25 C-50 Description Functional groups Weakly basic Diethylaminoethyl anion exchanger Strongly basic Diethyl-(2-hydroxyanion exchanger propyl)aminoethyl Weakly acidic Carboxymethyl cation exchanger Strongly acidic Sulphopropyl cation exchanger Counter ion Chloride Chloride Sodium Sodium

Sc k trao i ion
(Ion- exchange chromatography - IEC) X l (vi Sephadex v Sepharose acid v base ch c dng nng 0,1M): 1. Khuy nh nha trong ~ 5V th tch trng vi 0,5 M HCl (anionit) hoc 0,5 M NaOH (cationit), 30 pht to phng, thnh thong khuy. 2. Ra sch bng nc (bng phu lc ht chn khng), hoc bng giy lc cho n khi pH t hn 4 (sau acid) hoc thp hn 8 (sau kim) 3. Khuy nh nha trong 5V 0,5 M NaOH (anionit) hoc 0,5 M HCl. 30 pht, thnh thong khuy. 4. Lp li bc 2 n y nha c loi ht protein cn ln, v cc ion proton hoc hydroxyl lin kt yu vi cc nhm tch in ca cc cationit hoc anionit, chng c th c thay th d dng bng cc ion tng ng ca m c la chn.

Sc k trao i ion
(Ion- exchange chromatography - IEC)

Chn m:
pH cn trong khong cho php ca nha v cn khng gy hi n protein.

protein c th gn c ln nha, t nht pH phi cch 0,5 hoc tt hn l 1 n v so vi pI ca n (cao hn i vi anionit, thp hn vi cationit). pH cch qu xa s to ra s lin kt qu mnh vi nguy c gy bin tnh v hiu sut thp.

Sc k trao i ion
(Ion- exchange chromatography - IEC)

Cn bng m: nha cn cn bng hon ton trong m trc khi dng

1. Cho m x l vo 1 lng tng ng ca m c nng cao gp 10 ln m thng, trn v 30 pht to phng. 2. pH c th b thay i, khuy nh nha v chnh li pH.

3. nha 30 pht, ra li trn phu vi m dc gp 5 ln.

4. Nhi v ra ct vi m thng cho n khi dch chy ra t n pH cn.

Sc k trao i ion
(Ion- exchange chromatography - IEC)

Chun b ct:
Thng t l gia ng knh v chiu cao ct t 1/10 n 1/20. Lng nha cn dng c tnh t dung lng trao i ca nha v lng mu th ri thng ly tha ra 50%. Nha c cho (nhi) vo ct, y c bng thu tinh

Sc k trao i ion
(Ion- exchange chromatography - IEC)

Cho mu v chit rt:

Mu cn thm tch k i m chy trc khi cho ln ct, th tch khng quan trng (c th nhiu t bt k). Ra u tin vi t nht 2 ln (thng l 5 ln hoc hn) th tch ct loi ht protein khng bm.

Sau protein gn trn ct c y ra bng cch: 1. tng lc ion ca m 2. thay di pH ca n (h thp vi anionit, tng ln vi cationit) 3. hoc c hai bin php trn.
Thng ngi ta dng cch u v iu khin c cn thn hn. Lc ion cun cng thng l 1 i vi a s protein.

Sc k trao i ion
(Ion- exchange chromatography - IEC)

Hoc nng m c th tng, hoc c gi nguyn v tng cc ion khc (nh NaCl). Cch sau hay hn v kh nng m, do pH, c gi nguyn trong qu trnh tch

Nha c th bo qun 40 oC vi cht dit khun (0,03% toluene), vi cationit c th dng azid.

Sc k trao i ion
(Ion- exchange chromatography - IEC) Resolution in ion exchange chromatography
The result of an ion exchange experiment (as with any other chromatographic separation) is often expressed as the resolution (Rs) between the peaks of interest

Determination of the resolution (Rs) between two peaks

Sc k trao i ion
(Ion- exchange chromatography - IEC)

Separation results with different resolutions

Sc k trao i ion
(Ion- exchange chromatography - IEC)

Hypothetical chromatogram
V0 = void volume, VR1 = elution volume for peak 1, VR2 = elution volume for peak 2, Vt = total volume, Vb1 = peak width for peak 1, Vb2= peak width for peak 2.

Sc k trao i ion
(Ion- exchange chromatography - IEC)

The net charge of protein as a function of pH