Affinity Chromatography:

Theory and Practice

Renate Scheibe --- SS 2012

Anatomy Biotechnology

Biochemistry (in vitro) Physiology (in vivo)

Structure Purification

Enzymology Cell biology

Solvent Gel matrix

Cell components Substrates, effectors

α

Side chains (at Cα-atom) of the 20 proteinogenic amino acids:

Protein structure

Quaternary structure

+ -

+ -

+ -

+ -

+ -

+ -

+ -

+ - +

-

+ - +

- + -

+ -

+ -

+ -

+ -

+ -

+ -

+ -

+ - +

- + -

+ -

+ -

+ -

+ -

+ -

Surface of a typical protein

= hydrophobic patch

Monomeric proteins: 12-25% hydrophobic aa

Oligomeric proteins: 28-38% hydrophobic aa

Interactions between and within macromolecules

1. Van der Waals forces (attraction between atoms)

2. Ionic forces (salt bridges) A-….B+

3. Hydrogen bonds

4. Hydrophobic interactions (driven by entropy of the total system:

macromolecule + H2O)

5. Covalent bonds (e.g. disulfide bridges)

… O H O

1 covalent bond corresponds to 20 non-covalent bonds .

Example: Globin Heme in hemoglobin: More than 60 non-covalent interactions

Between organic molecules there are week, but many interactions:

Chromatography

E.g. gel permeation, ion exchange, hydrophobic interaction, affinity

Detection: Enzyme activity Immunological methods

Size Exclusion Chromatography (SEC) (= Gel permeation or Gel filtration)

Ideally without any adsorption of the proteins to the matrix

Adsorption chromatography

Ion exchange

Chromatofocusing

Adsorption to anorganic gels (HA etc.)

Hydrophobic chromatography (HIC)

Reversed Phase chromatography (RP)

Metal chelate chromatography

Covalent chromatography

Affinity chromatography

Dissociation constant

Attraction between ligand/matrix and soluble protein/compound

Kp = m · p

q

m = binding sites

p = free protein

q = bound protein

Langmuir Isotherm-Plot:

Partition coefficient

α =

q = bound protein

pt = free + bound protein

qpt

Example:

α2 = 0.4

α1 = 0

Concentration of FREE protein (p)

% s

ite

satu

rati

on

q

m

t

% s

ite

satu

rati

on

q

m

t

Total amount of protein (pt)

Ion Exchange Chromatography

Ionenaustausch

+

-

= Tris-H+

= Cl-

= negativ geladenes Protein

= Ionenaustauscher (z.B. DEAE-Sephadex)

3 4 5 6 7 8 9 pH

+

-

IEP

Bindung an Kationenaustauscher

Stabilitätsbereich des Enzyms

Bindung an Anionenaustauscher

Net

tola

du

ng

de

s P

rote

ins

Vorversuch zur Ermittlung des pH-Wertes:

4,5 5,0 5,5 6,0 6,5 7,0

Puffer + Enzym Ionenaustauscher

Optimierung der Bindebedingungen:

Hydrophobic chromatography

+

= unstructured water = structured water

= Butyl-, Pentyl-, Hexyl-, Octyl-, etc. Phenyl-, Naphtoyl-, Trityl-

Elution by decrease of ionic strength

and decrease of temperature

addition of organic solvent

addition of polyols (ethylene glycol)

addition of non-ionic detergent

decrease or increase of pH

entropy-driven interaction

Affinity chromatography

Affinity chromatography

+ Washing

Adsorbed sample

Elution

+

+

Matrix

Spacer

Ligand

Affinity in biological systems (specific binding)

Lectin – Glycoprotein

Enzyme– Substrate

Enzyme– Inhibitor

Hormone– Receptor

Transport protein – Substrate

Nucleic acid – Protein

Biotin – Avidin

Antigen – Antibody

Protein – Protein

Binding and/or elution can be specific or unspecific

Bioselective elution from ion exchangers

- - - - -

+ -

-

-

-

+ +

+ +

- +

+ -

+

-

+

- - - - -

CO

O-

CO

O-

Ligand

+ -

-

-

-

+

+ +

+

+ -

-

-

Na+

CO

O-

CO

O-

Ligand +

- - - - -

Na+ Na+ Na+ Na+ Na+

Elution with ligand

Enzyme-ligand: Net charge -1

Enzyme: Net charge +1

Electrostatic attraction

Electrostatic repulsion

Desorption from immunoadsorbents: (Interaction = hydrophobic as well as electrostatic forces)

1) Change of pH (e.g. pH 2.2 oder pH 11)

2) Decrease of polarity (e.g. with dioxan 10% or ethylene glycol 50%)

3) Denaturing with urea or guanidinium-HCl

4) Chaotropic ions (at pH 6-8):

Cl- < J- < ClO4- < CF3COO- < SCN- < CCl3COO-

(up to 3 M)

5) Additional options:

- Electrophoresis

- H2O

Purification of biological molecules

Immobilisation of a binding partner ( +/- Spacer)

Affinity adsorption of the other partner

Elution a) with chaotropic salt

b) with soluble ligand

c) with another binding substance

N

NN

N

NH

O

HOH OH

H

CH2 O P O

OH

O-

HSepharose 4B-NH-(CH2)6

5‘-AMP

5‘ AMP-Sepharose 4B

N

NN

N

NH

O

HO OH

H

CH2 O P O

OH

O-

H

POH O

O-

Sepharose 4B-NH-(CH2)6

N

NN

N

NH2

O

HO OH

H

CH2 O P O

O

O-

POH O

O-

P O

O

OHO

CH2

H HOH OH

N+

CO NH2

2‘ 5‘ ADP

2‘ 5‘ ADP-Sepharose 4B

NADP+

Nucleotide analoga as affinity ligands

O

O-

CH3

NH3

+

CH2

O

O-NH3

+

NH2

+

O

O-

CH3

NH CO

CH3

NH3

+

1

2

3

Immobilisation of alanine

..and of AMP

O

OH OH

N

NN

N

NH2

OCH2P

O

OH OH

N

NN

N

NH

OCH2P

O

NH

N

NN

N

NH2

OCH2

OH

P

N6-acyl AMP C8-acyl AMP Ribose-attached AMP

Matrix

Spacer

Ligand

a) Activation of the matrix b) Immobilisation of the spacer c) Immobilisation of the ligand by covalent coupling

Preparation of

Coupling reaction (0.01 M NaOH, 60°C, 2 h, + NaCl)

N

N

N

Cl

HO + N

N

N

O

Triazine dyes Cibacron Blue 3GA (a) and Procion Red HE-3B (b)

Reactive dye

Metal chelating chromatography (IMAC)

Apart from Ni2+, the following metal ions can be used: Co2+, Cu2+, Zn2+ etc.

NTA = nitrilo triacetic acid

Interaction between His-tag and Ni-NTA

Strep-tag Sequence: AWRHPQFGG

Covalent chromatography

SH + HS Enz S-S Enz HS Enz Elution + RSH

-SH : Glutathione, I-thio-2,3-hydroxypropane, Cysteine, Cystamine

Hg-OH + HS Enz Hg-S Enz HS Enz Elution + RSH

-Hg-OH : p-amino-phenyl-mercuri-acetate p-chloro-mercuri-benzoic acid p-hydroxy-mercuri-benzoic acid mersalyl

Weitere Anwendung: Zur Herstellung von Affinitätschromatographie-Medien bzw. aktivierten Medien zur Immobilisierung weiterer Liganden.

Beispiele für Affinitätschromatographie (1)

Immobilized ligand Protein to be purified Elution

- spez. Antikörper - Antigen Protein A

- entspr. Antigen - IgG

unspez. (chaotrope R.) unspez. ( „ )

Lektine ( z.B. Con A) Zucker Glykoprotein

Glykoproteine, Polysaccharide Lektine

Spez. Zucker Spez. Zucker oder unspez.

Avidin Calmodulin Phosphat oligo(dT)8 / Acriflavin Fluphenazin Arginin, Lysin 5‘-AMP 2‘5‘-ADP

Carboxylasen u.a. biotinhaltige Enz. Calmodulin-abh. E., z.B. Kinasen, Phosphodiesterase Phosphatasen, Kinasen Nukleotide u. Nukleinsäuren (z.B. mRNA) Calmodulin Proteasen NAD- und ATP-abh. Enzyme NADP-abh. Enzyme

unspez. spez. mit Ca2+ komplexierenden R. (EGTA) unspez. oder Substrat unspez. EGTA unspez. oder Arginin spez. oder unspez. spez. oder unspez.

Beispiele für Affinitätschromatographie (2)

Immobilized ligand Protein to be purified Elution

Heparin DNA- u. RNA-Polymerase unspez. (Salz) (=Nukleinsäure- Analogon) DNA-Ligase, Reverse Transkriptase (=Antikoagulans) Serumproteine Benzamidin Serinprotease ssDNA Repressoren, Histone GSH GST-Fusionsprotein (rekombinant) GSH Streptavidin „Strep-tag“-Peptid (rekombinant) Biotin Amylase Maltose-Bindeprotein Maltose (Fusionsprotein)

NH2

NH

NH2

Biospecific chromatography with macroligands:

Problems: 1) Unspecific interactions with Spacer or Matrix

2) Loss of biospecifity upon immobilisation of the ligand 3) Steric hindrance

Factors that can affect the affinity - Temperature (temperature ↑: affinity↓) - pH - Ionic strength (optimal between 50 und 200 mM) - Flow rate - Concentration of immobilised ligand

David S. Waugh: TiBiotech 23 (2005)

Pull-down approach (Pierce)

Bimolecular complementation

More methods based on affinities

Nature 2000 The yeast interactome

20-30% of the cell is occupied by macromolecules

the total concentration of protein and RNA inside an E. coli cell –

300-400 gl-1 (for proteins: 200-300 gl-1 , for RNA: 75-150 gl-1)

properties of macromolecules in solutions are studied at 1-10 gl-1 or less

The crowded state of the cytoplasm in eukaryotic (left) and E. coli (right) cells Each square represents a cube face of 100 nm length (Ellis, 2001)

Supplemental material

Sepharose

Agarose

Sephadex

Dextran, quervernetzt mit Epichlorhydrin

ClCH2 CH

CH2

O

Sephacryl

Acrylamid

Sepharose

bzw. Ultrogel AcA (Acrylamid/Agarose) oder Trisacryl GF CH CH2

O

NH

CH2OH

CH2OH

CH2OH

n

C

O

NH2 + GA C

O

NH CH

(CH2)3

CHO

(CH2)2 CH

CHO

C

CHO

(CH2)2 CHO CH CH

CHO

NH R

+ NH2-R

+ Ligand: z.B. + Glutaraldehyd:

OH OH

+ CNBr C

O

O

NH + O C N

OH

O

NH2

+

NH R

Cycl. Imidocarbonat Isoharnstoff

+ NH2-R

z.B. + Bromcyan:

z.B. + Bisepoxiran:

OH

CH2 CH

O

CH CH2

O

+ CH2 CH CH CH2

OOH

O

+ NH2-R

CH

OH

CH2 NH R

epoxi-aktivierte Matrix

a) Aktivierung der Matrix:

z.B. + Carbonyldiimidazol (CDI):

OH

NN

NN

C

O

+ NN

C

O

O+ NH2-R

NHC

O

O R

Reacti-Gel

O

O

CH2OH

OH

OH

O

+ NaJO4

O

O

CH2OH

O

O

O

+ NH2-R + NaBH4

O

O

CH2OH

N

O

N

R R

O

O

CH2OH

O

NOH OH

R

+

z.B. + Perjodat:

z.B. + Tosylchlorid:

OH

S

O

O

Cl CH3+ S

O

O

O CH3+ NH2-R

RNH

b) Immobilisieren eines Spacers Häufig verwendete Spacer-Arme: 6-aminocapronsäure (ACA) Ethylendiamin (EDA) Poly-Glycin Poly-L-Lysin Diaminodipropylamin (DADPA)

c) Kovalente Kopplung von Spacer-Matrix mit Liganden (mit Kondensationsreagentien):

OH

O + EEDQ

OH

O C2H5

O

O

NH R

Gemischtes Anhydrid

+ NH2-R

OH

O CNR N R1

+ O

O C N

NH R1

R

Isoharnstoffester

+ NH2-R O

NH R

Carbodiimid