Expression VectorExpression of cloned genes produces large quantities of protein

Components of expression vector

1. replication origin2. polylingker (MRS or MCS)3. Selective marker4. promoter5. operator6. ribosome binding site7. gene encoding repressor

그림 19.7 외부 DNA 절편은 제한효소를 이용하여 플라스미드 내로 삽입될 수 있다 .

pET26-preCGT7379 bp

kan

f1 origin

lacI

preCGT

NcoI (6676)

XhoI (7218)

HindIII (5873)

HindIII (6887)

NdeI (5071)

NdeI (5851)pET26b

53 6 0 bp

kan

f1 or ig in

lacI

Bam H I (5159 )

Eco RI (516 5)

H in dIII ( 518 4 )

Nco I (513 7 )

Nde I (50 7 1)

Xho I (519 9 )

pET vector

pET26

pET26

Regulation of Protein Expression in pET System

• Double induction by IPTG– T7 RNA polymerase (98 kDa)– target gene (only in T7lac vectors)

• Compatible with a wide range of expression hosts– requires DE3 lysogen

그림 11.1B Lac operon 의 조절

Procedure to purify proteins fused with an affinity tag

1. Fusion of GST gene to target protein gene (C- or N-terminal)2. Expression in recombinant strain3. Cell disruption to prepare cell extract4. Binding of the target proteins to resins via the affinity interac-tion between affinity tag(GST) and ligand (glutathione)

Electrophoresis

Cross-linked polymerpolyacrylamide

acts as a molecular sieve, slowing the migrationof proteins approximately in proportion to theircharge-to-mass ratio.

SDS-polyacrylamide gel

SDSCH3(CH2)11SO4

-

Na+

Purification of RNA polymerize from E. coli

gel stained with a protein-specific dye (e.g. coomasie blue)

Expression of K6UbGLP-1 in recombinant E. coli

Protein Purification via Ion Exchange Chromatography

Protein......

Specific Binding Site

Charged group- Asp, Glu, Lys, Arg, His

Size, Shape

Hydrophobic patch- Phe, Trp, Ile, Leu, Val etc

Metal chelating group- His, Trp, Cys

Use of chromatography• Production of biopharmaceuticals

Photograph courtesy of Phar-madule AB

Pilot and large-scale production

of Biopharmaceuticals

GE Healthcare Bio-Sciences

supplies proven integrated

solutions for process

chromatography

What happens in chromatography?

• Molecules to be separated diffuse into the beads

• They bind under one set of condi-tions and are re-leased under (usu-ally) other condi-tions

• Different molecules interact differently

Liquid-filled gel

bead

Column Gel

Separation principles in chromatographic purifi-cation

Gel filtration

Size

HIC (hydrophobic interaction)

Hydrophobicity

Ion exchange

Charge

Affinity

Biorecognition

Reversed phase

Hydrophobicity

05/nov/02

What is ion exchange chromatography?

Ion exchange chromatography is a form of LC that separates molecules on the basis of their charge

Useful at all stages of purification and at all scales

Controllable

High selectivity, high capacity

Concentrating, high recovery

• Interaction between opposite charges• Charged groups on the proteins interact with charged

groups on the ion exchanger. • Different proteins have different charges and interact

differently.• Anion or cation exchange• When the protein is negatively charged, it is an anion -

anion exchange• When it is positively charged, it is a cation - cation ex-

change

Separation by charge

• Some of the charged re-

gions which will influence

ion exchange

• Different proteins have

different charges and dif-

ferent patterns of sur-

face charge

Basis for selectivity

NH3R

COOH+

NH3R

COO+

-

R NH2

COO-

Low pHPositive charge

High pHNegative charge

Hydrogen gained

Hydrogen lost

Effect of pH on charge

Overa

ll c

harg

e o

n p

rote

in

-

+

NH3R

COOH+

NH3R

COO+

-R NH2

COO-

acid isoelectric point alkalineexcess positive charge balanced positive and negative charge excess negative charge

The overall charge on a protein depends on pH

pH3 10

Titration curves

Charg

e o

n p

rote

in

-

+

pH3 10

Anion exchanger

Cation exchanger

Controlling selectivity by pH

Ion-Exchange Chromatography

Example: Cation-exchange chromatography

Lane M: Marker proteinsLane 1: E. coli cells before inductionLane 2: E. coli cells after induction with IPTGLane 3: Soluble fraction after cell disruptionLane 4: Soluble fraction after heat treatmentLane 5: Anion exchange chromatographyLane 6: Cation exchange chromatography

Purification of Taq DNA polymerase expressed in recombinant E. coli