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