7/29/2019 Purificacion Proteinas.pdf

1/24

Protein Purification

Principles and Methods

7/29/2019 Purificacion Proteinas.pdf

2/24

Proteins

Complex, polymeric, asymmetric and sensitive molecules

Contain covalent bound prosthetic groups and non-covalentbound cofactors

Many non-covalent bounds e.g. Hydrogen-Bounds, Dipol-

Interactions and Hydrophobic-Interactions

Weak interactions are important for structure and function

(activity) of the protein

In most cases the purification must be gentle!

7/29/2019 Purificacion Proteinas.pdf

3/24

Before the purification

Cultivation of bacteria

Cell disruption: Periplasmic and cytoplasmic proteins are released

Centrifugation leads to a soluble fraction (supernatant) which containsall soluble periplasmic and cytoplasmic proteins and a membranefraction from which membrane bound proteins can be solubilised withdetergents (e.g. Triton X-100)

The soluble or membrane fraction are the start point of the furtherpurification by chromatography

Cell disruption: French Press

Lysozyme

Ultrasonic

7/29/2019 Purificacion Proteinas.pdf

4/24

French Press

7/29/2019 Purificacion Proteinas.pdf

5/24

Membrane Proteins

Peripheral membrane proteins: in most cases soluble in buffers withhigh or low ionic strength or high pH

Integral membrane proteins: they contain trans membrane helices andmust be solubilised to conserve conformation and function of theprotein

7/29/2019 Purificacion Proteinas.pdf

6/24

Solubilisation of integral membrane proteins

Solubilisation of proteins is done with a detergents concentration abovethe CMC to ensure the incorporation of membrane lipid into detergentmicelles.

CMC = critical micelle concentration

depends on temperature, ionic strength and pH of the buffer andconcentration of uncharged substances like urea or alcohol

7/29/2019 Purificacion Proteinas.pdf

7/24

Some detergents

Ionic detergents:

Sodium-Dodecylsulfate: denatures Proteins ( SDS-PAGE)

Na-Deoxycholate: preticipates by pH

7/29/2019 Purificacion Proteinas.pdf

8/24

Which proteins are purified?

Metabolic pathways

Energy production

Aim: biochemical characterisation (Reactivity, subunit composition,organic and inorganic cofactors, 3D structure)

and why?

7/29/2019 Purificacion Proteinas.pdf

9/24

Purification strategies

Protein stabilisation:

Integral membrane Proteins: Solubilisation

Purification at 4C: reduces protease activity

Addition of protease inhibitors: commonly used are EDTA andPMSF (toxic)

Quickly load on first column after cell disruption and ultracentrifuge

Main impurities are removed first, lesser in the second or third step

Max 5% impurities are acceptable

In general: Max 4 purification steps

No steps with purification factor < 5

No steps with < 30% yield

No steps which last longer than one day and one night

7/29/2019 Purificacion Proteinas.pdf

10/24

Chromatography

Separation material in columns, streamed by buffer

liquid chromatography

HPLC: high pressure/performance liquid chromatographyFPLC: fast protein liquid chromatography

7/29/2019 Purificacion Proteinas.pdf

11/24

FPLC unit

7/29/2019 Purificacion Proteinas.pdf

12/24

Separation principles

Size:

size exclusion chromatography (= gel filtration, = gel permeationchromatography)

Charge

anion or cation exchange chromatography; chromatofocusing

Hydrophobicityhydrophobic interaction chromatography (HIC)

Affinity

affinity chromatography

Solubility

ammonium sulfate precipitation (non chromatographic, rel. imprecise)

7/29/2019 Purificacion Proteinas.pdf

13/24

Size exclusion chromatography

Different pore sizes, depending on the size of the proteins

Separation is based in diffusion slow flow rate

Pressure sensitive materials

7/29/2019 Purificacion Proteinas.pdf

14/24

Example for a separation by gel filtration

7/29/2019 Purificacion Proteinas.pdf

15/24

Noteworthy about chromatography

Gel filtration:

limited sample volume: 2-3 mllimited flow rate gel filtraion takes timedelution of the sample by a factor of about 3

low purificatopn factor: 3-6needs column buffer with high ionic strength: min 0.1 M

Ion exchage chromatography:

To remember: Protein binding depents on electrostatic interactions withthe column material.Strength of binding depents on pH and ionic strength ofthe buffer, the pI of the protein and the charge density onthe column.

In general: Technical easier than gel filtration: columns could bepacked at the FPLC.

Sample volume can be multiple times the column volume.Higher flow rates.Purification factor: 3-15

Sample gets concentrated. Dont use charged detergents!

7/29/2019 Purificacion Proteinas.pdf

16/24

Ion exchage chromatography

Anion exchanger Cation exchanger

7/29/2019 Purificacion Proteinas.pdf

17/24

Binding behaviour of proteins

pI = isoelectric point of the protein the pH value at which the possesno net charge

The pI determines the charge of the protein at a given pH

pH > pI negative net charge

pH < pI positive net charge

7/29/2019 Purificacion Proteinas.pdf

18/24

pI and protein separation on ion exchange columns

7/29/2019 Purificacion Proteinas.pdf

19/24

But...

pI is not alone responsible for the binding behaviour of proteins

Binding is sometimes influenced by local charges and not by the net

charge of the protein

7/29/2019 Purificacion Proteinas.pdf

20/24

Example for a separation by ion exchange chromatography

7/29/2019 Purificacion Proteinas.pdf

21/24

7/29/2019 Purificacion Proteinas.pdf

22/24

Carrier materials

Poly sugars

Sepharose (Agarose), Sephadex (Dextran), Sephacel (Cellulose)

Rare sugars can hardly be utilised by bacteria.

Low flow rates Material changes its Volume depending on the ionic strangth

Material settles over time

Beads Polystyrol/Divinylbenzen beads with charge carrier added

Relative high flow rates

Good pressure stability, no compression

Hard charges could stress the protein

7/29/2019 Purificacion Proteinas.pdf

23/24

Beads and tentacle

Charges are linked with flexible spacers to polymeric beads. This leads to asoft binding of proteins despite of hard charges on the matrix

Relative high flow rates Good pressure stability

High capaticity

Perfusion beads Porous material, beads with chanels

Very big surface

Highly pressure stable

Very high flow rates

7/29/2019 Purificacion Proteinas.pdf

24/24