chap 9. ion exchange chromatography -...
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Research & Development
Upstream Processes
Production
Downstream Processes
Product
Gene Discovery
Cloning & Transformation
Transformation
Cell Line Development
Media Preparation
Microbial Fermentation
Mammalian Cell Culture
Harvest Cells
Cell Disruption
Protein Purification
Analytical Tests
Protein production flowchart
Chap 9. Ion Exchange Chromatography
Protein Separation and Purification
Why Purification? to understand the structure and functions of proteins
Purification Procedure : 1. Crude extract 2. Subcellular fractionation
3. Fractionation of proteins---- Size, Charge, pH,
Solubility, Salt concentration, Dialysis
Methods of Protein Purification and Identification:
1. Column Chromatography ---- Ion exchange chromatography
Size-exclusion chromatography
Affinity & hydrophobic chromatography
2. Gel Electrophoresis ------- SDS gel electrophoresis
Isoelectric focusing
Two dimensional electrophoresis
(purification)
(Identification)
Harvest Cells
Cell Disruption
Total Proteins
Pure Protein
Analytical Tests
Growth Medium
Cell Debris
Unwanted Proteins
Centrifugation
Filtration
Enzymatic
Chemical
Physical
Purification Steps
Protein purification protocol
→ Chromatography
Chromatography
• Separation based on relative affinities of compound
for solid phase and liquid phase
• Ion exchange chromatography
• Gel filtration chromatography
• Affinity chromatography
• High performance liquid chromatography (HPLC)
Fast protein liquid chromatography (FPLC)
분류 종류
고정상의 특성에 따라 - Column Chromatography
- Planar Chromatography
고정상, 이동상의 극성에 따라
- Normal phase chromatography
: 극성이 높은 silica gel, alumina, 종이 등을 고정상,
극성이 낮은 유기용매를 이동상으로 사용
- Reversed phase chromatography
: 고정상으로 극성이 낮은 탄화수소(C18 또는 C8)를
이동상으로 극성이 높은 수용액을 사용
분자 분리하는 방법에 따라
- Affinity Chromatography <- by affinity
- Gel filtration Chromatography <– by size
- Ion exchange Chromatography <– by charge
이동상의 특성에 따라 - Gas Chromatography
- Liquid Chromatography
Column Chromatography
Ion Exchange Chromatography
Ion exchange chromatography makes use of electrostatic
properties of the protein of interests.
exchanger
Effect of the chemical environment on pKa
** The pKa of any functional groups is greatly affected by its chemical environment.
Similar effects can be observed in the active site of enzymes.
Titration Curve of Amino Acid
1. First COOH group titrated,
then NH3 group
2. Tow buffer zones
3. Amino acid is amphipatic
4. Isoelectric point (pI)
5. Below pI → positive charge,
6. Above pI → negative charge
Ion Exchange Chromatography
immobile bead composed of a
positively charged
“ion exchange resin”
+ +
+
+
+
+ +
+
+
+
Surrounded by a mobile counter ion
e.g., Cl-
Protein
sample
Negatively charged
Neutral
Positively charged
Ion Exchange Chromatography
+ +
+
+
+
+ +
+
+
+
Negatively charged proteins will
displace the negatively charged
counter ions
+ +
+
+
+
+ +
+
+
+
• Electrostatic properties of a protein determine the type of ion exchange resins it interacts with. In principle:
– Protein is positively charged if solution pH < pI; It should bind to negatively charged resins, or cation exchanger;
– Protein is negatively charged if solution pH > pI; It should bind to positively charged resins, or anion exchanger;
– Note that in practice, protein surface has local charges that may different from total charge of the protein.
• Examples of cation exchangers include : carboxymethyl (CM) and sulfopropyl (SP)
• Examples of anion exchangers include: diethylaminoethyl (DEAE), quaternary amine (QAE)
protein
surface - -
- + +
+ -
nonpolar
nonpolar
Beads are charged
• Cation exchange resins are
negatively charged
– Positively charged amino
acids will stick
• Anion exchange resins are
positively charged
– Negatively charged amino
acids will stick
• Charged polymers are usually immobilized through
covalent bonds on insoluble matrix, such as cellulose or
polyacrylamide. The protein of opposite charge become
bound to the matrix while other proteins flow through the
column. After washing, the protein bound to the matrix can
be eluted by adding salts.
UV
absorb
ance
Increasing salt concentration
protein B
Protein B
protein B
Protein B
Protein A
ProteinANa+ SO3+--CM
Fraction eluted from the column
[ protein ]
Protein can be differentially removed from the
column by adding salt
[ salt ]
Load proteins
on column
Positively charged
or neutral proteins
0 0
[ protein ]
Protein can be differentially removed from the
column by adding salt
[ salt ]
Load proteins
on column
0
Fraction eluted from the column
0
Negatively charged
proteins
weak strong
[ protein ]
Test all the fractions for activity
[ salt ]
Load proteins
on column
0
Fraction eluted from the column
0
Most active
• Sodium counterions replaced by charged amino acids
• Increasing gradient of sodium knocks off amino acids in order of increasing affinity
Separation of Complex Mixtures
Amino acid composition
using ion exchange HPLC