purification of enzyme

7/30/2019 Purification of Enzyme

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PURIFICATION OF ENZYME

It is important to study it for the understanding

of structure, mechanism, kinetics, regulation

& role in complex system.

Also for the industrial & medical purposes theenzyme purification is extremely important.

For the medical purposes the highly pure

enzymes are needed.


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A CASE STUDY OF ENZYME

PURIFICATION

Purpose study of particular enzymepurification procedure from the thousands ofclosely similar enzymes.

Idea of selection of different techniques forthe purification of particular enzyme from theparticular organism because seeminglysimilar enzyme in two different species maybe different & so their purification techniquesmay be different.


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THEE. coli BACTERIA

E. coli are the mostextensively studied exampleof the bacteria.

The various strains ofE. coliare extensively studied at

genetic level. Most studied strain is E. coli

K12.

Many eukaryotic geneshave been cloned into E. coli

& expressed.


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E. coli RNA POLYMERASE


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DISCOVERY OF RNA

POLYMERASE The RNA polymerase was

first isolated & purified bytwo scientists named SamWeiss & Jerard Hurvitz.

The mechanism of RNA

synthesis by RNApolymerase wasdiscovered by SeveroOchoa & he was awardedNobel Prize for Physiology& Medicine with Arthur

Kornberg.


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INFORMATION REGARDING RNA

POLYMERASE OFE. coli

subunit Size(aa) Size (kd) Gene

Alpha () 329 36511 rpoA

Beta () 1342 150616 rpoB

Beta () 1407 155159 rpoC

Sigma () 613 70263 rpoD

Omega () 91 10237 rpoZ


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PROCEDURE OF RNA

POLYMERASE PURIFICATIONThe purification procedure is distributed in some few steps.

They are as follow. Growth of cells in media & harvesting them by

centrifugation.

Breaking open the cells by proper technique.

Centrifugation step to remove cell debris & ribosomes.

Fractionation & re-extraction by appropriate salt.

Re-dissolution of gained precipitates & ion exchangechromatography step.

Again ion-exchange chromatography step by phospho-cellulose as ion-exchanger.

Gel filtration chromatography step. Checking of purity of enzyme.


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STEP 1- GROWTH OF CELLS &

HARVESTING BY CENTRIFUGATION

The E. coli K12 strain isgrown in particular

broth medium.

Centrifugation at low

speed

Gaining of about 75% of

maximum growth after

incubation period.


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STEP 2- BREAKING OPEN THE

CELLS BY PROPER TECHNIQUE

Blending at high speed with glass beads

Adding of deoxyribonuclease

Filtration


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STEP 3- CENTRIFUGATION TO REMOVE

CELL DEBRIS & RIBOSOMES

Addition ofdeoxyribonucleasecleaves the DNA &reduces the viscosity ofthe medium.

The ribosomes are free in

the prokaryotic cells & sothey can be removed bythe centrifugation at highspeed.

Here the centrifugal field is100000g & time for run is 2

hours.


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STEP 4 - FRACTIONATION & RE-

EXTRACTION BY APPROPRIATE SALT

The supernatant in previous step.

Fractionation by (NH4)2SO4 of 33 50%

saturation.

Re-extraction with (NH4)2SO4 of 42%saturation.


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STEP 5 - RE-DISSOLUTION OF PRECIPITATES

& ION-EXCHANGE CHROMATOGRAPHY.

Re-dissolve the precipitates into proper buffer

system.

Loading onto the DEAE-cellulose ion-exchangechromatography column.

Elution with KCl gradient.


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DEAE-CELLULOSE


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DEAE-CELLULOSE ION EXCHANGECHROMATOGRAPHY


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STEP 6ION EXCHANGE

CHROMATOGRAPHY BY PHOSPHO-

CELLULOSE

Ion-exchange chromatography on

phosphocellulose.

Elution with 0.35 mol/dm3 KCl.


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STEP 7GEL FILTRATION

CHROMATOGRAPHY STEP

Bio-gel A Gel filtration chromatography

It is cross-linked agarose gel.

Column length is 1.5m.

Bio-gel A fractionates in the Mr range 10000to 2000000.

This whole procedure gives overall yield ofenzyme 54 mg per 200 gm of frozen cells.

56% of activity can be gained by thisprocedure.


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STEP 8 - CHECKING OF PURITY

OF ENZYME

Ultracentrifugation (only single peak is obtained)

Electrophoresis in 8 mol/dm3 urea (the four

bands correspond to the correct amounts of thefour subunits of different size)


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PURIFICATION FACTORS OF

DIFFERENT STEPS

Definition

The

purification

factor of step

is defined asthe increase

in specific

activity after

that step.

STEP PURIFICATION

FACTOR

THREE 1.9

FOUR 4.4

FIVE 5.9

SIX 5.1

SEVEN 1.1


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PURIFICATION OF RNA POLYMERASE BY

GENETIC METHOD

Useful for the understanding of the

interactions of RNA polymerase with the

DNA.

Use of T7 RNA polymerase T7 promotersystem.

Different method Different reagents


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T7 RNA POLYMERASE

T7 bacteriophage genome encodes an enzyme RNApolymerase.

Transcription from 53 direction.

Requires Mg2+ for the optimal activity.

Does not affected by the rifampicin antibiotic.

Molecular weight is 99 kd.

They have very much affinity towards theirpromoters. So the transcription efficiency is very highin comparison with the other RNA polymerases.

It can be stimulated by BSA & spermidin.


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STEPS INVOLVED IN OVEREXPRESSION &

RECONSTITUTION OF RNA POLYMERASE

SUBUNITES

High level expression of alpha, beta, beta &sigma subunits into the E. colicells.

Cell disruption by sonication & recovery ofsubunits in form of insoluble inclusion bodies.

Dissolution of inclusion bodies of beta, beta &sigma in the high concentrations of denaturantslike urea or guanidinium chloride.


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MORE RAPID PROCEDURE FOR

RECONSTITUTION OF THE INTACT

ENZYME

Recombinant alpha subunit is produced in a

histidine tagged form.

Mixing of these tagged subunit with crude

preparation of the other subunits.

Metal affinity chromatography on an immobilized

nickel matrix (Ni2+-NTA-agarose)

Elution with 5mmol/dm3 imidazol.


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REFERENCES

Introduction to enzymology by Stevens.

Internet- www.nobelprize.org

Google image search

Broth, centrifuge, deoxyrebonuclease, glassbead blender, ultracentrifuge, (NH4)2SO4extraction, DEAE-cellulose ion-exchangechromatography, bio-gel A gel filtration

chromatography, T7 RNA polymerase-T7promoter system.
http://www.nobelprize.org/http://www.nobelprize.org/


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THANK YOU

purification of enzyme

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