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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