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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
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