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 the enzyme purification is extremely important. For the medical purposes the highly pure enzymes are needed.

A CASE STUDY OF ENZYME PURIFICATION

Purpose study of particular enzyme

purification procedure from the thousands of closely similar enzymes. Idea of selection of different techniques for the purification of particular enzyme from the particular organism because seemingly similar enzyme in two different species may be different & so their purification techniques may be different.

THE E. coli BACTERIA

E. coli are the most

extensively studied example of the bacteria. The various strains of E. coli are extensively studied at genetic level. Most studied strain is E. coli K12. Many eukaryotic genes have been cloned into E. coli & expressed.

E. coli RNA POLYMERASE

DISCOVERY OF RNA POLYMERASE

The RNA polymerase was

first isolated & purified by two scientists named Sam Weiss & Jerard Hurvitz. The mechanism of RNA synthesis by RNA polymerase was discovered by Severo Ochoa & he was awarded Nobel Prize for Physiology & Medicine with Arthur Kornberg.

INFORMATION REGARDING RNA POLYMERASE OF E. coli

subunit Alpha () Beta () Beta () Size(aa) 329 1342 1407 Size (kd) 36511 150616 155159 Gene rpoA rpoB rpoC

Sigma ()
Omega ()

613
91

70263
10237

rpoD
rpoZ

PROCEDURE OF RNA POLYMERASE PURIFICATION

The 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 exchange chromatography step. Again ion-exchange chromatography step by phosphocellulose as ion-exchanger. Gel filtration chromatography step. Checking of purity of enzyme.

STEP 1- GROWTH OF CELLS & HARVESTING BY CENTRIFUGATION

The E. coli K12 strain is

grown in particular broth medium. Centrifugation at low speed Gaining of about 75% of maximum growth after incubation period.

STEP 2- BREAKING OPEN THE CELLS BY PROPER TECHNIQUE

Blending at high speed with glass beads

Adding of deoxyribonuclease
Filtration

STEP 3- CENTRIFUGATION TO REMOVE CELL DEBRIS & RIBOSOMES

Addition of

deoxyribonuclease cleaves the DNA & reduces the viscosity of the medium. The ribosomes are free in the prokaryotic cells & so they can be removed by the centrifugation at high speed. Here the centrifugal field is 100000g & time for run is 2 hours.

STEP 4 - FRACTIONATION & REEXTRACTION BY APPROPRIATE SALT

The supernatant in previous step.

Fractionation by (NH4)2SO4 of 33 50%

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

EFFECT OF (NH4)2SO4 ON PROTEIN FRACTIONATION

STEP 5 - RE-DISSOLUTION OF PRECIPITATES & ION-EXCHANGE CHROMATOGRAPHY.

Re-dissolve the precipitates into proper buffer

system. Loading onto the DEAE-cellulose ion-exchange chromatography column. Elution with KCl gradient.

DEAE-CELLULOSE

DEAE-CELLULOSE ION EXCHANGE CHROMATOGRAPHY

STEP 6 ION EXCHANGE CHROMATOGRAPHY BY PHOSPHOCELLULOSE

Ion-exchange chromatography on

phosphocellulose. Elution with 0.35 mol/dm3 KCl.

STEP 7 GEL 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 10000 to 2000000. This whole procedure gives overall yield of enzyme 54 mg per 200 gm of frozen cells. 56% of activity can be gained by this procedure.

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 the four subunits of different size)

PURIFICATION FACTORS OF DIFFERENT STEPS

Definition

STEP THREE FOUR FIVE

The purification factor of step is defined as the increase in specific activity after that step.

PURIFICATION FACTOR 1.9 4.4 5.9

SIX
SEVEN

5.1
1.1

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 promoter system. Different method Different reagents

T7 RNA POLYMERASE
T7 bacteriophage genome encodes an enzyme RNA

polymerase. 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 their promoters. So the transcription efficiency is very high in comparison with the other RNA polymerases. It can be stimulated by BSA & spermidin.

T7 PROMOTER SYSTEM

STEPS INVOLVED IN OVEREXPRESSION & RECONSTITUTION OF RNA POLYMERASE SUBUNITES

High level expression of alpha, beta, beta &

sigma subunits into the E. coli cells. Cell disruption by sonication & recovery of subunits in form of insoluble inclusion bodies. Dissolution of inclusion bodies of beta, beta & sigma in the high concentrations of denaturants like urea or guanidinium chloride.

CONTINUED
Recovery of alpha subunit in a native form

either in the soluble fraction after cell lysis or salt extraction of insoluble material. Reconstitution of core enzyme by mixing the subunits (alpha, beta & beta) in the presence of denaturing agents like 6 mol/dm3 urea or guanidinium chloride. Denaturant removing by dialysis.

CONTINUED
Adding of 10-20 & (v/v) glycerol & salt like

0.2-0.3 M KCl & maintain the protein concentration below about 0.5 mg/ml. Ion exchange chromatography to separate core enzyme complex from free subunits. Adding of a small 2-4 fold molar excess of sigma subunit to prepare intact enzyme.

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.

REFERENCES

Introduction to enzymology by Stevens. Internet- www.nobelprize.org Google image search Broth, centrifuge, deoxyrebonuclease, glass bead blender, ultracentrifuge, (NH4)2SO4 extraction, DEAE-cellulose ion-exchange chromatography, bio-gel A gel filtration chromatography, T7 RNA polymerase-T7 promoter system.

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