Download ppt - Protein Purification Sac Ky

Bốn kỹ thuật sắc ký (chromatographic techniques)

1.Sắc ký trao đổi ion [Ion Exchange Chromatography (Charge specific)]

- Hydroxyapatite-Salt tolerant ligands

2. Sắc ký lọc gel [Size Exclusion (Gel Filtration) Chromatography] - Size/shape dependent

3. Sắc ký ái lực [Affinity Chromatography (Biological Interaction)]– Protein A (Protein G and others)– IMAC (His/surface specific)– Specific affinity resins (lectines, dyes…)– Immunospecific media

4. Sắc ký tương tác kỵ nước [Hydrophobic Interaction Chromatography (Hydrophobic Patches)]- Mixed Mode- Hydrophobic charge induced chromatography

What does „strategy“ mean?

Combination of different types of chromatography• Due to the complexity of mixtures of biological materials, often severalsteps are necessary to produce a homogeneous protein.• Successive separation steps must be synchronized optimally to avoidunnecessary re-buffering or dialysis steps.• Otherwise the recovery of the biological activity and the mass recovery,which both depend on the number of purification steps, will be reduced.• For example, starting with a high salt concentration in HIC is in contrastto ion exchange, where one has to start with very low ionic strength.

Lưu ý trong sắc ký

• Giữ được hoạt tính sinh học:– Không sử dụng dung môi có pH quá cao hoặc thấp,– Nồng độ muối thích hợp,– Thời gian tách ngắn để tránh tối thiểu quá trình phân hủy protein– Nhiệt độ thấp

Basic Considerations• Cell disruption method• Homogeneity of the source material• Stability of the target protein• pH• Buffer (Stabilisators)• Temperature (4°C)• Proteinases (proteolysis, rapid decrease of biol. activity)• Main contaminating compounds• Refolding?

Protein Purification Tutorial Intro –table of common techniques

Affinity chromatography

binding to small molecules

Ion exhange chromatography

Isoelectricpoint(charge)

Size-exclusion chromotography

Size / shape

MethodsProperty

B¶ng cc ph ¬ng php th êng dïng ®Ó tinh chÕprotein

solubility Precipitation with ammonium sulfate (salting out)*

* KÕt tña ammonium sulfate lµ rÊt rÎ, dÔkiÕm vµ phï hîp cho cc mÉu lín. Nãth ênglµ b íc®Çutiªn trongs¬ ®å tinhchÕ.

• Cc b íctinhchÕ cã xu thÕ b¶o ®¶m cho protein ë tr¹ng thi tù nhiÖn. MÆcdï thÕmétsèprotein cã thÓ ti tù nhiÕn(re-natured), hÇuhÕtlµ kh«ngthÓ!

• §Ó tinhchÕprotein tronghçnhîp, cãrÊtnhiÒuph ¬ngphp khc nhauthÝchhîpchotõnglo¹i protein. Chóng khc nhau vÒ:

– TÝnhbÕnnhiÖt*

* ChohÇuhÕtcc qu tr×nhtinhchÕprotein, tÊt c¶ cc b íc thùc hiÖn ë ~5°C ®Ó lµm gi¶n sùbÊtho¹t.

Protein Purification Tutorial Intro side bar

H×nh ¶nh gel protein ví i c c ® êng ch¹y chØ ra tr×nhtùc c b í c tinh s¹ch

60060,00010080Ion exchange

80000

100000

Ho¹t tÝnh(unit)

20040090Size-exclusion

10100001400Crude cellular extract

Ho¹t tÝnh riªngUnit/mg

Protein tæng(mg)

ThÓ tÝch ph©n ®o¹n (ml)

Çc b í c

B¶ng tinh chÕ

Add row with ammonium sulfate data. Include two columsat end called Purification factor and Yield.

Purification factor

Yield

100%

20 80%

3 75%

1

Note: The type and order of steps are customized for each protein to be purified. An effective purification step results in a high yield (minimal loss of enzyme activity) and large purification factor (large increase in specific activity).

Protein Purification Tutorial Intro – flow chart. Purification is a multi step procedure

MÉuKü thuËt t ch

Fractionation

TinhchÕlµ métc«ng®o¹n cãnhiÒub í c.

Is there activity?Set aside No CombineFractions

yesMonitor purity

Assay total protein

Assay enzyme activity

Pure?

Prepare for analytical technique

yes

No

LËp l¹i ví i c c kü thuËt kh c ®Õn tËn

khi tinh khiÕt

Protein Purification Tutorial Intro – first steps.

• First steps– 1. Nguån. Métnguåntètlµ ph¶i rÎ

vµ s½n cã. NhiÒuprotein cã nhiÒu trong c c m« ®Æc biÖt, vÝdônh hemoglobin trongm u. V× lýdo nµy mµ nhiÒum« lµ nh÷ngnguåntuyÖtvêi choprotein cñachóngta.

– 2. ThönghiÖm: hÇu hÕt c c thö nghiÖm lµ c c ph¶n øng ho hâcóc t c nhê c c ho¹t tÝnh ®Æc tr ng. Çc protein kh«ng cã ho¹t tÝnh th êng® î c thö nghiÖm nhêSDS polyacrylamidegel.

Protein Purification Tutorial Assay – develop an assay

• First steps – Ph t triÓnthönghiÖm

1. Mét thönghiÖmchométenzyme lµ métph ¬ngph p ®Ó x c ®Þnhho¹t tÝnhcñanã.

TõthönghiÖmnµy ® î c lËp l¹i nhiÒulÇn,nã rÊt quan träng nh ng nã lµ b í c ®¬n gi¶n. Th«ngth êngho¹t tÝnh enzyme ® î chiÓnthÞsùthay ®æi vÒsùhÊpthômµ cãthÓ®o ® î cnhêspectrophotometer. VÝ dôthö ribonuclease®o sù thay ®æi vÒ sù hÊp thô ®i cï ng ví i sù ph©n huû RNA thµnh c c ribonucleotide.

Protein Purification Tutorial Intro – Preparing the sample (Crude Extract)

Protein tõ c c tÕbµo hoÆc m«

First steps: ChuÈn bÞ mÉu– triÕt th«.

TÕ bµo vi sinh vËthoÆcm«

Ph vì tÕbµo,m« hoÆc c¬ quan

NghiÒn, ®ångho , Siªu ©m,¸p lùc,thÈm thÊu KÕt l¾ng cã c c tÕ bµo, organelles,

mµngvµ c c protein mµng

N í cnæi ví iprotein hoµ tan

Protein Purification Tutorial Segway to Chromotography

• L u ý: Ví i môc ®Ých ®Ó t ch ®ñ l î ngprotein, chóngtacÇnl î ngm« ban ®Çuhµngkilogram. Khèi l î ng nµy tèt nhÊt cho viÖc sö dông kÕt tña (nh .kÕt tñaammonium sulfate). Sau®ã tinhchÕ, c c cét lí ncãthÓsödôngtõgram ®Õnmilligram. Khèi l î ng®Ó chay gel lµ microgram.

Protein Purification Tutorial Intro – flow chart. Purification is a multi step procedure

Sample Separationtechnique

Fractionation

TinhchÕlµ métc«ng®o¹n gåmnhiÒub í c.

Is there activity?Set aside No CombineFractions

yesMonitor purity

Assay total protein

Assay enzyme activity

Pure?

Prepare for analytical technique

yes

No

Repeat with another separation

technique until pure

Protein Purification Tutorial Over view of the apparatus

– Cét thuûtinh(Glass column)– B×nhchøa(Reservoir)– ChÊtnhåi r¾n (Solid matrix – beads)– Dung dÞchprotein (Solution of

protein).– Effluent.– Other terms?

S¾c kýcét (Column Chromotography) – tr ênghî pchung

We think it would be better to have a resevoir attached to a cap on the top of the column via a plastic tube, show minimal liquid on top of the column bed, show a tube coming out of the bottom of the column that has a clamp of stopcock. Perhaps a schematic on the left and a photo of an actual column running in a cold box over a fraction collector on the right.

Should emphasize that the basic set=up is the same for all column types, but the characteristics of the beads vary.

Protein Purification Tutorial Load sample (4 protein mix)

• H×nh ¶nh hån hî pprotein n»m ë trªn bÒ mÆt

DÞchchiÕt th« ® î c®Ó trªn ®ØnhcñachÊtnhåi r¾n (solid matrix). (trongtr ênghî pnµy chóngtasödônghçnhî p4 protein, hiÓnthÞb»ngc c mµu kh c nhau.)

• (As the animation proceeds)Protein chuyÓn ®éng ë c c tèc ®é kh c

nhau qua chÊt nhåi dùa trªn c c tÝnh chÊt cñaprotein vµ kiÓu chÊt nhåi cét.

Note - this should be shown as a single band (possibly brown or striped with four colors)

Protein Purification Tutorial Collect fractions.

• Sù ph©n ®o¹n • V× cét t chprotein ë d¹ng hån hî p, “effluent” nháxuèngc c èngph©n ®o¹n mµ nãchuyÓn®éngví i métvËntèc®Æctr ng. Çc èngnµy gäi lµ c c ph©n ®o¹n (fraction).

ë ®©y chóng ta cã20 èng. M¸y thuph©n ®o¹n ë hÇuhÕtc c phßngthÝnghiÖmth êngcãkho¶ng 75-200 èng.

Be sure to remove color from column as it drips into the tubes below! If the sample is spread over three tubes, the center tube will be darker in color.

Protein Purification Tutorial --3 questions—

• Trongthùc tÕ, protein kh«ng cã mµu v× vËy chóng ta sÏ cã3 c©u hái:

1. Lµm thÕ nµo chóng ta biÕt ® î c ph©n ®o¹n cã protein?

2. Ph©n ®o¹n nµo cã protein mongmuèn?

3. Chóngta®nhgi¸ ®é tinhkhiÕtnh thÕnµo?

???

Protein Purification Tutorial Question 1 – take A280 Screen 1.

• Tæng sèprotein cãthÓdù®o n nhê

®o sùhÊpthôtrªn m y so mµu ë 280

nm. Çc amino acid th¬m hÊp thô

¸nh s ng ë b í c sãng lµm cho tÊt c¶

protein cã hÊp thô ë 280nm. NhiÒu

m y ph©n ®o¹n ®o A280 khi ch¹y cét

.

Question 1. Lµm thÕnµo chóng ta biÕt® î cph©n ®o¹n cãprotein?

Take A280

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20Fraction#

Protein Purification Tutorial Question 1 – take A280 Screen 2 .

• Tæng sèprotein cã thÓ dù®o n nhê®o sù hÊp thô trªn m y so mµu ë 280 nm. Çc amino acid th¬m hÊp thô¸nhs ngë b í c sãnglµm cho tÊtc¶ protein cã hÊp thôë 280nm. NhiÒum y ph©n ®o¹n ®o A280 khi ch¹y cét

• Çcgi¸ trÞ cã thÓ dùng®åthÞsoví i sèph©n ®o¹ngäi lµ elution profile.


A280

Plot values

02520025852000252000

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20Fraction#


• Protein tæng cã thÓ dù ®o n nhê ®o hÊp thô ë 280 nm trong m y so mµu.

• Çc gi¸ trÞcãthÓdùng®å thÞso ví isèph©n ®o¹n gäi lµ elution profile.

• L u ý c c peak trªn ®å thÞ. NãhiÓnthÞë ph©n ®o¹n nµo cãprotein.


A28002520025852000252000

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20Fraction#

A280

Fraction #

Peaks


• Total protein a can be estimated by taking the absorbance at 280 nm in a spectrophotometer.

• The values can be plotted against the fraction number in is what is called an elution profile.

• Notice the peaks on the graph. These indicate where the fractions are that contain protein.

Question 1. How do we know which fractions contain protein?

A28002520025852000252000

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20Fraction#

A280

Fraction #

Peaks


• Ho¹t tÝnh enzyme cã thÓ thÓ x c ®Þnh nhê thùc hiÖn thö enzyme trªn mçiph©n ®o¹n mµ cãprotein.

Question 2. Ph©n ®o¹n nµo cãprotein mongmuèn?

A28002520025852000252000

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20Fraction#

A280

Fraction #

Enz. Assay.

Fraction#


• Ho¹t tÝnh enzyme cã thÓ x c ®Þnh nhê thùc hiÖn thö nghiÖm enzyme trªn mçi ph©n ®¹on cã protein.

• L u ý c c kÕt qu¶ thö nghiÖm enzyme. Ho¹t tÝnhcaonhÊt t ¬ngøngví i 1 trongc c peak.

Question 2. Ph©n ®o¹n nµocãproteinmong muèn?

A28002520025852000252000

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20Fraction#

A280

Fraction #

EnzAssayResults

Need to substitute values for the colored spots since we are switching to an absorbance based assay.

B©y giê chóng ta cã thÓ bá c c èng mµ kh«ng cã enzyme.


• Enzyme activity can be determined by performing an enzyme assay on each fraction that contains protein.

• L u ý c c kÕt qu¶ cña thö enzyme. Ho¹t tÝnhcaonhÊt t ¬ng® ¬ngví imét trong c c peak.

• Lo¹i bá c c ph©n ®o¹n kh«ng cã protein.

Question 2. Ph©n ®o¹n nµo cãprotein mong muèn?

A28002520025852000252000

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20Fraction#

A280

Fraction #

EnzAssayResults

Protein Purification Tutorial POOL fractions – screen 1

KÕt hî p c c ph©n ®o¹n cã ho¹t tÝnh.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20Fraction#

Fraction #

Pool fractions

1. TiÕp theo chóng ta gom c c ph©n ®o¹n cã ho¹t tÝnh enzyme.

It may be useful to consider more than just the activity of a fraction. Specific activity is a measure of the amount of enzyme activity per amount of protein (units/mg). The higher the specific activity, the higher the purity. When pooling fractions, judgement is needed as to whether to optimize yield or specific activity.

Protein Purification Tutorial POOL fractions – screen 2

Gom c c ph©n ®o¹n cã protein vµ ho¹t tÝnh.

Fraction #

1. Çc ph©n ®o¹n ® î c gom l¹i ví i nhau.

How do we monitor the progress of the purification?

Protein Purification Tutorial Assess purity – screen 1

1. Xemtrªn gel. NÕu chØcãmétband, nãcãthÓtinhkhiÕtvµ lµ métmonomer, homo-dimer, hoÆchomo-multimer. NÕucãnhiÒuband th× sùtinhchÕcãthÓch a s¹ch hoÆc c c subunit cñaenzyme.

2. TÝnhho¹t ®é riªngth«ngqua x c ®Þnh ho¹t tÝnhenzyme/protein tæng.

Question 3: MÉu gom®ã cãtinhkhiÕt kh«ng?

Standards | Crude Ext. | Pooled fractions

80000

100000

Activity

(units)

20040090Separation method 1


Specific activity

Units/mg

Total Prot

(mg)

Fraction vol

(ml)

Procedure

Purification Table

Purification Table

Results: 1. Gel shows more than one band.

Since the sample is not pure, you must pass pooled sample through another separation technique

Another separation

Protein Purification Tutorial Assess purity – screen 2

1. Quans t nãtrªn gel. Monomer sÏ cãoneband.

2. TÝnh ho¹t tÝnh riªng nhêtûsèho¹t tÝnhenzyme tæng/ tængsèprotein.

Question 3: mÉuenzyme tinhchÕcãtinhkhiÕthay kh«ng?

Standards | Crude Ext. | Pooled fractions

60000

80000

100000

H¹t tÝnh tæng(units)




Ho¹t tÝnhriªngUnits/mg

Protein tæng(mg)

ThÓ tÝch ph©n ®o¹n (ml)

B í c thùc hiÖn

Purification Table

Results: 1. Gel shows one band2. Specific activity is 15000. Looks good

Your protein seems pure. YOU’RE DONE!!!.

Protein Purification Tutorial Intro – table of common techniques

S¾c ký i lùcKÕt hî p ví i c c ph©n tö nhá

S¾c ký trao®æi ion§ iÓm®ång®¼ng®iÖn(®iÖntÝch)

S¾c ký läc gelKÝch th í c

Ph ¬ng ph pTÝnh chÊt

B¶ng c c ph ¬ng ph p th«ng dông tinh chÕ protein

TÝnh tan KÕttñab»ngammonium sulfate (salting out)*

Ammonium sulfate precipitation is cheap, easy, and accommodates large sample sizes. It is commonly one of the first steps in a purification scheme.

Protein Purification Tutorial Gel filtration 1 -basis

• S¾c kýcét läcgel t chprotein dùatrªn kÝchth í c.

• Chóng ta sÏ b¾t ®Çu ví i 4 protein.• Chóng ta muèn tinh chÕ“yellow one”

60 Kd

Low pI (6)

20 Kd

Low pI (7)

20 Kd

Medium pI (7)

5 Kd

Hi pI (8)

Here’s our sample mix of proteins.Our goal is to purify protein #….

Protein Purification Tutorial Gel filtration 2 - close up of beads

• C¬ sëchÝnhcñaküthuËts¾c ký cét läcgel lµ lç rçngcñachÊtnhåi cét.

Run column

Need two pore sizes (other size bigger than black proteins, smaller than existing pores.)

Protein Purification Tutorial Gel filtration 3 - run close up of column

• Gi¸ thÓcñacét läc gel lµ c c h¹t cã lç.

• Çc protein lí n xanh kh«ng võa ví i c c lç v× vËy nã chÈy nhanh h¬n.

• Çc protein trung b×nh red/yellow bÞ gi÷ l¹i trong c c lç.

• Çc protein nháblack bÞ gi÷ l¹i trong c c lç l©u h¬n.

We’re wondering how this will work in animation. The black can permeate all pores and the space between beads, the yellow and red can permeate the space between beads and larger pores, the green will be restricted to the space between beads.

Protein Purification Tutorial Gel filtration 4 - zoom out

Click on the peak that represents the protein of the largest molecular weight?

Tubes march in from left A280

Fraction #

Be sure to keep in mind that the colors will either be in the column or in the tubes, not both!

Protein Purification Tutorial Gel Filtration 5.

• • Many columns are commercially made. Here are some examples.

Fig 1.1. Scanning electron micrographof an agarose gel. Magnification x 50,000.Ref. Anders S. Medin,PhD Thesis, Uppsala University 1995.

Sephadex./

This could be moved to the earlier view of the porous beads.

Protein Purification Tutorial ION –EXCHANGE 1

• S¾c kýcét trao®æi ion t chprotein dùatrªn ®iÖntÝch.

• Chóng ta b¾t ®Çu ví i 4 protein.• pH 7.2• Cét tÝch ®iÖn d ¬ng

60 Kd

Low pI (6)

20 Kd

Low pI (7)

20 Kd

Medium pI (7)

5 Kd

Hi pI (8)

Need to include a slide on how to determine the charge on a protein, given its pI and the pH.

Protein Purification Tutorial Ion Exchange 2 – loaded proteins

• Gi thÓ cña trao ®æi ion cã tÝnh ®iÖn d ¬ng.

• ChóngtanghÜc i g× sÏ xÈy ra?

pos

pos

pos

pos

pos

pos

Run column

Protein Purification Tutorial Ion Exchange 3 –column run

• Gi¸ thÓ cña trao®æi ioncã tÝnh®iÖnd ¬ng.

• ChØc c protein tÝch®iÖnd ¬ngch¹y qua cét tÝch®iÖnd ¬ng. Çc protein kh c bÞgi÷ l¹i trongcét.

pos

pos

pos

pos

pos

pos

These beads are porous, too, so you can show the proteins moving right through the beads in the animation, ifyou like.

Protein Purification Tutorial Ion Exchange 4- zoom out

ChØ cã c c protein tÝch ®iÖn d ¬ng ch¹y qua cét.

Lµm thÕ nµo ®Ó chóng ta lÊy ® î c c c protein kh c?

Tubes march in from left

A280

Fraction #

120

Protein Purification Tutorial Ion Exchange 5 - zoom out

T¨ngnång®é muèi.


Add salt

A280

Fraction #

Protein Purification Tutorial Ion Exchange 6 – after salt

T¨ngnéng®é muèi.Protein nµo sÏ rakhái cétsau®ã?


-

A280

Fraction #

- ---

Add salt

Don’t show the charges on the color spots -students should figure this out on their own!


T¨ngnång®é muèi.Protein nµo sÏ ®i ra tr í c?


---

A280

Fraction #

+

---

Run column


§ á vµ vµngcãcï ng®iÖntÝch(net charge) vµ sÏ ® î c röacï ngnhau (co-elute).


A280

Fraction #

Salt concentration

1.5

0.0

Protein Purification Tutorial Ion Exchange 9 – Increase salt conc. Again.

T¨ng nång ®é muèi


A280

Fraction #

Salt concentration

1.5

0.0

Add salt

Protein Purification Tutorial Ion Exchange 10 – rum column prompt.

Run the column.


A280

Fraction #

Salt concentration

1.5

0.0

Run column

Protein Purification Tutorial Ion Exchange 11 – results.

Run the column.


A280

Fraction #

Salt concentration

1.5

0.0

Run column

Protein Purification Tutorial Ion Exchange 12 – results.

L u ý r»ng 2 protein ®i ra cï ng mét thêi gian. T¹i sao?

Protein cñachóngtacãtinhkhiÕt? ChóngtamuèntinhchÕred one

.


A280

Fraction #

Salt concentration

1.5

0.0

Protein Purification Tutorial Affinity Chromotography.

• Affinity Chromotography • See notes below

Protein Purification Tutorial Monitoring progress.

• Monitoring progress. • ChóngtacÇnmétsèth«ngtin trªn SDS –PAGE vµ ho¹t tÝnh riªng.

Place final table here - we can include the thing plot with protein conc going down, enzyme amount remaining constant, and specific activity onthe rise.