Protein Purification Strategies

8/6/2019 Protein Purification Strategies

1/28

Protein Purification

Tutorial

Minnie MurugesanDr. Scotts Group

07-12-05


2/28

Eventful Protein Purification

Grow cells in media (vector+tag) Centrifuge, Collect the pellet

Lyse the cells (appropriate buffer)

Purification Strategy

Pilot ExpressionSDS PAGE, Assay

Solubility

Aggregation

Recombination

Characterization

Mass Spectroscopy

X-rayCrystallography

Functional Assay


3/28

OUTLINE

Chromatography techniques AffinityChromatography (AC)

Hydrophobic Interaction Chromatography (HIC)

Ion Exchange Chromatography (IEC)

Gel Filtration (GF)

Capillary electrochromatography (CEC)

Other New Strategies for Protein Purification

Solubility, Aggregation and Re-folding of Proteins

Invented by a Russian botanist named Mikhail Tswett in 1903. He separated

plant pigments using glass columns packed with calcium carbonate.


4/28

1. Evaluate an assay for the protein of interest

2. Shortlist a method to have a reasonable source for that activity

Protein Purification Strategies

(http://www5.amershambiosciences.com/aptrix/upp00919.nsf/Content/LabSep_EduC%5CAboutPurBiom%5CHowToCombine)


5/28

Three Phase Strategy

For Membrane Proteins

(www.amershambiosciences.com)


6/28

Chromatographic Mode Acronym Separation Principle

Non-interactive modes of liquid chromatography

Size-exclusion chromatography SEC Differences in molecular size

Slalom chromatography (forDNA) - Diff. in length and flexibility

Interactive modes of liquid chromatography

Ion-exchange chromatography IEC Electrostatic interactions

Normal-phase chromatography NPC Polar interactions

Reversed-phase chromtography RPC Dispersive interactions

Hydrophobic interactionchromatography

HIC Dispersive interactions

Affinity chromatography AC Biospecific interaction

Metal interaction chromatography MIC Complex w/ an immobilized metal

Chromatographic Modes of Protein Purification

(Christian G.Huber, BiopolymerChromatography,Encylcopedia in analytical chemistry, 2000)


7/28

AffinityChromatography

Surface bound with

Epoxy, aldehyde or aryl ester groups

Metal Interaction Chromatography

Surface bound with

Iminodiacetic acid + Ni2+/Zn2+/Co2+

Affinity Chromatography



8/28

Metal Interaction Chromatography (AC)

Points to Note:1. Avoid chelating agents

2. Increasing incubation time

3. Slow gradient elution

(www.qiagen.com)


9/28

Affinity Chromatography

Binding Capacity (mg/ml) medium

12mg of histag proteins (MW= 27kDa)

Depends on Molecular weight

Degree of substitution /ml medium

~15Qmol Ni2+

Backpressure ~43psi

Change the guard column filter



10/28

Biopolymer(phenyl agarose - Binding Surface)

Driving force for hydrophobic adsorptionWater molecules surround the analyte and the binding

surface.

When a hydrophobic region of a biopolymer binds to

the surface of a mildly hydrophobic stationary phase,

hydrophilic water molecules are effectively released

from the surrounding hydrophobic areas causing a

thermodynamically favorable change in entropy.

Temperatureplays a strong role

Ammonium sulfate, by virtue of its good salting-out properties and high solubility in water is used as

an eluting buffer

Hydrophobic Interaction Chromatography

Hydrophobic region



11/28

Fractogel matrix is a methacrylate resin upon which polyelectrolyte

Chains (or tentacles) have been grafted. (Novagen)

Ion Exchange Chromatography

Globular

Protein

Deformation due to interaction

with conventional ion exchanger

Maintenance of conformation whileinteracting with tentacle ion exchanger

(www.novagen.com)


12/28

Gel Filtration

(http://lsvl.la.asu.edu/resources/mamajis/chromatography/chromatography.html)


13/28

Immuno Affinity Chromatography

(http://www.cellmigration.org/resource/discovery/discovery_proteomics_approaches.html)


14/28

ATP immobilized on polyacrylamide resin

DNA Binding Proteins

Heparin Sepharose

Negatively charged proteins (pI >7) are not captured/separated effectively.

(www.novagen.com)


15/28

Capillary Electrochromatography

CEC is an electrokinetic separation technique

Fused-silica capillaries packed with stationary phase

Separation based on electroosmotically driven flow

Higher selectivity due to the combination of chromatography

and electrophoresis

Fused silica tube filled with porous methacrylamide-stearyl

methacrylate-dimethyldiallyl ammonium chloride monolithic

polymers, 80 x 0.5mm i.d., 5.5kV. High Plate count ~ 400,000

Height Equivalent to a Theoretical Plate /Plate Count (HETP) H = L/N

number of plates N = 16(t/W)2

whereL = column length, t = retention time, and W = peak width at baseline

(http://www.capital-hplc.co.uk)


16/28


17/28

GSTBind Purification KitsHisBind Purification Kits

Magnetight Oligo d(T) Beads

MagPrep Streptavidin Beads

Protein A and Protein G Plus Agaroses

STag Purification KitsStreptavidin Agarose

T7Tag Affinity Purification KitProteoSpin CBED (Concentration, Buffer Exchange and Desalting) Maxi

Kit Effectively desalts and concentrates up to 8 mg of protein with an

efficient, easy-to-use protocol.(Norgen BiotekCorporation)

ProteoSpinDetergent Clean-up Micro Kit Provides a fast and effective

procedure to remove detergents including SDS, Triton X-100, CHAPS, NP-40

and Tween 20.

Commercially available protein purification kits

(http://www.emdbiosciences.com)


18/28

Schematic of a Multi-dimensional Separation System


19/28

Fast Protein Liquid Chromatograph (FPLC)

1

2

3

5

4

No air bubbles

(Priming) Use degassed buffers

Injector Module

Column Inlet

Detector

Fraction

Collector

(www.pharmacia.com)


20/28

Protein Analysis

(http://www.cellmigration.org/resource/discovery/discovery_proteomics_approaches.html)


21/28

Reagent Derivatization Detection

o-PhthaldialdeyhdePrecolumn/

PostcolumnFL, Ex 340nm/Em 400nm

Fluorescamine Precolumn/Postcolumn

FL, Ex 390nm/Em 490nm

Indocyanine greeen PrecolumnFL, Ex 765nm/

Em 820-840nm

Detection of Proteins byDerivatization with Higher Sensitivity

1000 times more sensitive than UV-Vis detection

(Christian G.Huber,Encylcopedia in analytical chemistry, 2000)


22/28

Solubility of a protein

Membrane proteins

1. Removal of unbroken cells from the cell lysate by low speed centrifugation

(20 min at 10,000 g).

2. Isolation of the membrane particles from the supernatant by

ultracentrifugation (60 min at >100 000 g).3. Washing of the membrane particle to remove all soluble proteins.

4. Solubilization of protein from the membrane particles by a mild detergent.

(detergent: protein ratio = 1:10)

5. Phosphate buffers(0.1M-0.5M), 5-50% glycerol helps.

Depends strongly on the composition of the lysis buffer.

Salt concentration

Freeze-thaw protocol

* Freeze quickly on dry ice and leave for 3 min.

* Thaw immediately at 42 C. Vortex vigorously to mix well.

* Repeat the two previous steps three more times (4 cycles in all).

(http://www.ls.huji.ac.il/~purification)


23/28

Protein Aggregation

(http://www.integritybio.com/protein%20aggregation.htm)

Numerous physicochemical stresses can induce protein aggregation:

Heat, pressure, pH, agitation, freeze-thawing, dehydration, heavy metals,

phenolic compounds, and denaturants.


24/28

Solubilization of Aggregated Proteins

Denaturation and Renaturation

Variables Good starting point

Buffer composition (pH, ionic strength) 50 mM Tris-HCl, pH 7.5

Incubation temperature 30C

Incubation time 60 min

Concentration of solubilzing agent 6 M guanidine-HCl or 8 M ureaTotal protein concentration 1-2 mg/ml

Re-folding of Proteins

The addition of a mixture of reduced and oxidized forms of low molecular

weight thiol reagent usually provides the appropriate redox potential to allowformation and reshuffling of disulfide bonds

(1-3 mM reduced thiol and a 5:1 to 1:1 ratio of reduced to oxidixed thiol)

The most commonly used are glutathione, cysteine and cysteamine.

(www.biovectra.com)


25/28

Polyethylene glycol

(PEG 3350)

0.1-0.4 g/L L-Arginine hydrochloride 0.4-0.8M

Nondenaturating

concentrations ofUrea

< 2.0 M K-Glutamate ~5M

Nondenaturating

concentrations Gdm/ClH

< 1.0 M Proline ~1M

Methylurea 1.5-2.5 M Glycerol 20-40 %

Ethylurea 1.0-2.0 M Sorbitol 20-30 %

Formamide 2.5-4.0 M Sucrose ~1M

Methylfomamide 2.0-4.0 M Trehalose ~1M

Acetamide 1.5-2.5 M TMAO

(trimethylamine N-oxide)

~1M

Ethanol Up to 25% Sulfo Betaine ~1M

Reagents used for Re-folding of proteins



26/28

n-Penthanol 1.0-10.0 mM Lauryl Maltoside 0.06 mg/ml

n-Hexanol 0.1-10.0 mM CETAB 0.6 mg/ml

Cyclohexanol 0.01-10.0 mM CHAPS 10-60 mM

Tris > 0.4 M Triton X-100 10 mM

Na2SO

4or K

2SO

40.4-0.6 M Dodecyl Maltoside 2.0-5.0 mM

Cyclodextrin 20-100 mM Sarkosyl 0.05-0.5 %

Reagents used for Re-folding of proteins (Continued)



27/28

6xHis Tagged Protein Detection Directly on the Gel (from Pierce)

E. colilysates expressing 6xHis-tagged proteins,stained withthe Pierce

6xHis Protein Tag Staining Kit

(www.piercenet.com)


28/28

(www.biotech.ou.edu)

University ofOklahoma

Recombinant Protein Solubility Prediction

Type (or cut and paste) your protein sequence below, click on the "Submit" button, and

the solubility probability ofyour protein will be calculated.

The statistical model predicts protein solubilityassuming the protein is being over-expressed in Escherichia coli.

The input protein sequence has a 73.4 percent chance ofinsolubility when

overexpressed in E. coli. - mbh8 NADH dehydrogenase subunit - integral

membrane protein (NP_579159)The input protein sequence has a 80.5 percent chance ofsolubility

when overexpressed in E. coli. - replication factor A related protein (NP_579749)

Date post:	08-Apr-2018
Category:	Documents
Upload:	lem-lara
View:	228 times
Download:	0 times

Protein Purification Strategies

Documents