Protein Analysis and Purification

Analytical SeparationsGel-electrophoresisIEF2D-gels

Preparative SeparationsVarious chromatographic methods

Total E. coli Proteins - 2-Dimensional Gel

Overview of Protein Purification

IntroductionProtein PropertiesTypes of SeparationsProtein Inactivation/Stabilization

Overexpression of Recombinant ProteinsPurification Tags/Affinity Handles

Protein PurificationObject: to separate a particular protein from all other proteins and cell components

There are many proteins (over 4300 genes in E. coli)

A given protein can be 0.001-20% of total protein

Other components:

nucleic acids, carbohydrates, lipids, small molecules

Enzymes are found in different states and locations:

soluble, insoluble, membrane bound, DNA bound,

in organelles, cytoplasmic, periplasmic, nuclear

Scale of Purification

Micro ng-g

Normal lab mg-100 mg

Large lab-scale gm

Pilot plant 10 g-kg

Industrial 100 tons

Most common in research lab

Amount & Purity Required for Different UsesUse Amount needed Purity Required

Immunology g-mg

Polyclonal antibodies High

Monoclonal antibodies Medium

Enzymology mg High (>95%)

Physical Properties mg-g High

Enzyme/protein Chemistry mg-g High

Structure g High

Research Enzymes g-mg Variable; must be free of key contaminants

Pharmaceutical g-kg Very high (>99.99%) free of bacterial endotoxin

Industrial Enzymes kg-ton Variable, often low

Study Question #1You are given a shoe box full of an assortment of small objects including:

Ping Pong balls

Sugar cubes

Paper clips

1/2” brass screws

Iron filings

1. List the properties of each of these components that might help you fractionate them.

2. Devise the most efficient method you can for getting pure paper clips.

20 Naturally-occurring Amino Acids

Acidic:

D, E, (C, Y)

Basic:

K, R, H

Hydrophobic:

I, L, V, W, F

Polar:

S, T, N, Q

Other:

G, A, M, P

Main Types of Molecular Interactions

Hydrogen Bonds

N H - - - - N N-H + N

low temperature high temperature

N H - - - - O C strength is very dependent on geometry donor acceptor and distance (2.6-3.1 A)

Hydrophobic Interactions (waxy residues: Ileu, Leu, Val, Phe, Trp)

high salt high temperature low salt

Ionic Interactions (charged residues:Asp- Glu- S- Lys+ Arg+ His+)

low ionic strength high ionic strength

-+ - +Cl- Na+

...

H2OH

HHH

H HHH

Variables that Affect Molecular Forces

Temperature

Ionic strength

Ion type

Polarity of solvent (dielectric constant)

pH

Protein Properties - Handles for FractionationSize (110 Da/amino acid residue)

smallest most proteins largest

Amino acids: 30 100 1,000 15,000

MW (kDa): 3.3 11 110 1,600

Multi-subunit complexes can contain 5-30 subunits

Shape

globular (sphere) asymmetric (cigar)

Effects frictional properties, effective radius, movement through poresCentrifuge Gel filtration

Sediments slower

Appears smaller

Elutes earlier

Appears larger

Protein Properties - Handles for FractionationNet chargeIonizable group pKa pH2 pH7 pH12C-terminal (COOH) 4.0 oooooooo----------------------------------------

Aspartate (COOH) 4.5 oooooooooo-------------------------------------

Glutamate (COOH) 4.6 ooooooooooo------------------------------------

Histidine (imidazole) 6.2 +++++++++++++oooooooooooooooooooo

N-terminal (amino) 7.3 +++++++++++++++oooooooooooooooooo

Cysteine (SH) 9.3 ooooooooooooooooooooooo-----------------

Tyrosine (phenol) 10.1 oooooooooooooooooooooooooo-------------

Lysine (amino) 10.4 ++++++++++++++++++++++++oooooooo

Arginine (guanido) 12.0 ++++++++++++++++++++++++++++++o

Isoelectric pointpI = pH where protein has zero net chargeTypical range of pI = 4-9

Charge distribution

++

++

-

--

-

uniform

++ +

+

-- - -

clusteredversus

Protein Properties-Handles for FractionationHydrophobicity Hydrophobic residues usually are buried internally

The number and distribution on the surface vary

Can use Hydrophobic Interaction Chromatography

Solubility Varies from barely soluble (<g/ml) to very soluble (>300 mg/ml)

Varies with pH, ionic strength/type, polarity of solvent, temperature

Least soluble at isoelectric point where there is least charge repulsion

Ligand and metal binding Affinity for cofactors, substrates, effector molecules, metals, DNA

When ligand is immobilized on a bead, you have an affinity bead

HHH

hydrophobic patch

Protein Properties-Handles for Fractionation

Reversible association e.g., E. coli RNA polymerase

Post-translational modifications Carbohydrates, lipids, phosphates, sulfates

Can be very useful purification handles

E.g.: Use of plant lectins to bind certain glycoproteins

Specific sequence or structure Precise geometric presentation of amino acids on surface of a protein

Epitope for binding to a specific antibody; use immunoaffinity column

Binding site for another protein; use protein affinity column

Monomer (0.3M NaCl) Dimer (0.05 M NaCl)

Separation Processes that can be Used to Fractionate Proteins

Separation Process Basis of SeparationPrecipitation ammonium sulfate solubility

polyethyleneimine (PEI) charge, size

isoelectric solubility, pI

Chromatography gel filtration (SEC) size, shape

ion exchange (IEX) charge, charge distribution

hydrophobic interaction(HIC) hydrophobicity

DNA affinity DNA binding site

immunoaffinity (IAC) specific epitope

chromatofocusing pI

Electrophoresis gel electrophoresis (PAGE) charge, size, shape

isoelectric focusing (IEF) pI

Centrifugation sucrose gradient size shape, density

Ultrafiltration ultrafiltration (UF) size, shape

Typical Protein Purification Scheme

Protein Inactivation/StabilizationBuffers Solution Components

Protein Sources for PurificationTraditional natural sources Bacteria, animal and plant tissue

Cloning recombinant proteins into overexpression vector/host systems for intracellular production (E. coli the most used)

In vitro protein synthesis Transcription/translation systems

Total E. coli Proteins - 2-Dimensional Gel

What You Can Learn from Amino Acid Sequence 1. Molecular weight of the polypeptide chain 2. Charge versus pH; Isoelectric point 3. Extinction coefficient 4. Hydrophobicity & membrane spanning regions 5. Potential modification sites 6. Conserved motifs that suggest cofactor affinity

What You Can’t Learn from Amino Acid Sequence

1. Function 2. 3-Dimensional structure; Shape 3. Multi-subunit features 4. Ammonium sulfate precipitation properties 5. Surface features (hydrophobic patches, charge

distribution, antigenic sites)

Conclusion: Protein Purification is still very empirical!

Engineering Proteins for Ease of Purification and Detection

Once you have a gene cloned and can over-express the protein, you can alter protein to improve the ease of purification or detection

You can fuse a tag to the N-or C- terminus of your protein

You can decide to remove the tag or not

Basic strategies

Add signal sequence that causes secretion into culture medium

Add protein that helps the protein refold and stay soluble

Add sequence that aids in precipitation

Add an affinity handle (by far the most used is the His-tag)

Add sequence that aids in detection

CSH Protein Course -Sigma32 Purification

MW A B C D E F G A/3 B/3 D/3

’

32

225

50

35

10 kDa