Protein Manipulation
김경규
(성균관대 의학과)
Overview of Biochemical Experiments (characterization)
Immunochemistry
Antibody production
Physicochemical characterization:
MW (electrophoresis=EP, mass), size (EP, gel-filtration,
centrifuge), purity (EP, chromatography), concentration
(Bradford assay, spectroscopy), PI: electrofocusing
(Partially)
Purified
biological
materials
Functional assay
Identification (2D EP, Western, mass), enzymatic assay &
kinetic study (EP, UV-Vis, fluorescence, isotope), P-P
binding assay (EP, pull-down, migration assay, ITC, Biacore,
gel-filtration, blotting), P-D binding assay (bandshift assay,
footprinting assay), Chemical modification
Structure study
2nd structure (CD, IR), 3rd structure (EM, X-ray, NMR),
distance (fluorescence), conformational change
(fluorescence, CD, UV-VIS, IR)
Why do you need proteins? (amount & purity)
1.How are you going to prepare the large amount of protein?
- from tissue or using a recombinant system.
1.How are you going to purify protein?
- using their biochemical and biophysical characters.
1.How are you going to maintain their function (activity): keep their fold
in native state.
What do you need to know?
-Protein, what is it?
-Structure & folding
-Biochemical and biophysical characters
-Function : binding & catalytic activity
-What factors affect the structure and function of protein?
Purpose?
Antibody
production
Function
study
Structure
study
Production
Synthesis
(protein or
peptide)
Recombinant
protein
Endogenous
protein
Purity
Partially
purified
Maintenance
Short term
storage
> 95 %
Medium
term
storage
Long term
storage
Protein Chemistry
Protein → Amino Acids
20 Amino Acids
Protein folding
Alanine 7.2%
Cysteine 1.9%
Aspartic acid 5.3%
Glutamic acid 6.3%
Phenylalanine 3.9%
Glycine 7.2%
Histidine 2.3%
Isoleucine 5.3% Valine
Lysine 5.9%
Leucine 9.1%
Methionine 2.2%
Asparagine 4.3%
Proline 5.2%
Glutamine 4.3%
Arginine 5.1%
Serine 6.8%
Threonine 5.9%
6.6%
Tryptophane 1.4%
Tyrosine 3.2%
Protein stability and activity
Factors affecting the stability and activity of
proteins: temperature, pH, protease, microbial
contamination, protein concentration, organic
solvents, buffer, salt etc. (denaturation,
preteolytic digestion, chemical modification,
adsorption)
Purpose?
Antibody
production
Function
study
Structure
study
Production
Synthesis
(protein or
peptide)
Recombinant
protein
Endogenous
protein
Purity
Partially
purified
Maintenance
Short term
storage
> 95 %
Medium
term
storage
Long term
storage
Expression systems
Heterologous Protein Expression (host, vector)
1. Bacteria: E. coli, Bacillus etc (plasmid)
2. Yeast: Saccharomyces cerevisiae, Pichia pastoris (plasmid)
3. Insect cell: Spodoptera frugiperda (Sf), Trichoplusia ni TN-368 (High 5)
(Baculovirus)
4. Animal cell: CHO cells, 293 cells etc (virus or plasmid)
5. In vitro translation: E. coli extract, wheat germ extract, rabbit recticulocyte
(transcription & translation, plasmid)
6. Plant
Type
Cost
Time
Yield
Folding
Comment
bacteria
1
1
2
5
Yeast
2
2
4
3
Insect cell
3
2
3
2
Animal cell
4(5)
5
5
1
In vitro
(cell free)
5(4)
1
1
2
Prokaryotic expression
- Why E. coli? Simple and inexpensive.
(total 50 % of E.coli proteins can be the heterologous proteins.
- Why other systems? expressed proteins might not be active (folding problem).
- Components
* Regulatory elements for transcription control (promoter)
=> strong, tightly regulated, induction
* Translation initiation and termination
=> RBS or Shine-Dalgarno sequence (must be located optimally
from the start codon)
=> TAA is preferred as a stop codon (less prone to read-through)
* MCS site for insertion
* Marker for selection
Ori
Marker
Prokaryotic expression:
low expression level or insoluble protein
- Vector and host selection
* protease-deficient host
* rare codon t-RNA
* secondary structure of RNA near translation initiation site
* copy numbers of plasmid
* promoter selection
- Expression condition:
* growth temperature
* inducer concentration
* secretion (concentration and disulfide bond),
* co-expression with chaperone
- Fusion protein (+detection and purification)
pET system (unique features)
- Tight control of basal expression level
(DE3, pLysS, pLysE)
- Host selection
- Various fusion tags for purification
(Novagen Catalogue)
(Novagen Catalogue)
Prokaryotic expression - procedures
Primer design
PCR
Purification of PCR product
Restriction enzyme treatment
Gel extraction of the restricted PCR product
Ligation of PCR product with digested vector
Transformation into BL21(DE3)
Colony selection
Culture & IPTG induction
Cell harvest
Expression test in SDS-PAGE
Purification
XhoI
NotI
HindIII
SalI
F1 origin
EcoRI
BamHI
NcoI
TEV site
KpnI
NheI
NdeI
NcoI
KanR
pVFT1L vector
5378bp
lacI
Insect cell expression
Sf9 cells
Plaques
Transfection
Virus purification
infection
(Modified from
Invitrogen Catalogue)
Expressed proteins
Sf9 cells transfected with b-gal
Mammalian cell expression system : CHO cells
YGI
100 ug
DHFR
5 ug
YPI
YPI
No NTPs
CHO
DHFR
-
Add MTX
(DHFR
inhibitor)
DHFR+
YGI+
YPI
100-500X DHFR
100-500X YGI
YPI
YPI
YPI
YPI
Grow cells and
collect
conditioned
serum free
media
Mammalian cell expression system: 293 or Hella
cells
YGI
Virus infection
or transfection
YPI
YPI
293
cells
Selection
By
marker
YPI
Colony
selection
YPI
YPI
YPI
YPI
YGI+
Grow cells and
collect
conditioned
serum free
media
In vitro translation (cell-free protein synthesis)
(Modified from
Invitrogen Catalogue)
Heterologous protein expression (host, vector)
1. Bacteria: E. coli, bacillus etc (plasmid)
2. Yeast: Saccharomyces cerevisiae, Pichia pastoris (plasmid)
3. Insect cell: Spodoptera frugiperda (Sf), Trichoplusia ni TN-368 (High 5)
(Baculovirus)
4. Animal cell: CHO cell, 293 cell etc (virus, plasmid)
5. In vitro translation: E. coli extract, Wheat germ extract, rabbit recticulocyte
(transcription & translation, plasmid)
6. Plant
Type
Cost
Time
Yield
folding
comment
bacteria
1
1
2
5
insoluble
Yeast
2
2
4
3
Insect cell
3
2
3
2
Glycosylation
issue
Animal cell
4(5)
5
5
1
Glycosylation
issue
In vitro
(cell free)
5(4)
1
1
2
Good for toxic
proteins
Purpose?
Antibody
production
Function
study
Structure
study
Production
Synthesis
(protein or
peptide)
Recombinant
protein
Endogenous
protein
Purity
Partially
purified
Maintenance
Short term
storage
> 95 %
Medium
term
storage
Long term
storage
Protein purification
References :
Protein purification , Robert Scopes, Springer-Verlag
Modern experimental biochemistry, Rodney Boyer, Benjamin Cummings
Protein purification techniques, Simon Roe, Oxford
GE healthcare catalogue
,.
General consideration for purification
1. Maximize the yield and minimize the cost and time
2. Separate proteins using their physicochemical
characters
3. Remove major impurity first
4. Use the most effective method first
5. Use the most expensive method last
6. Make your protein active
Protein handling:
1. Principle: keep protein active and stable
2. Factors: contamination, denaturation, preteolytic
digestion, chemical modification, adsorption
3. Methods:
- Mild condition (pH, temp. surface) – avoid the
denaturation
- Concentration (avoid low and high conc.)
- Stability and degradation (low temp., protease inhibitor,
sodium azide, short exposure to high temp.)
- Stabilizing agent such as glycerol
- Reducing condition
- No freeze & thaw
Protein Purification – general scheme
Protein overexpression or raw materials
Cell Paste
Cell disruption and debris separation
Crude extract
Pretreatment of samples or low-resolution chromatography
Partially purified (1st) protein
Protein separation using chromatography
Partially purified (2nd) protein
Purity < 95 %
Protein separation using high-resolution chromatography
Purified protein
Function studies
Chromatography
-
-
Principle : samples can be interact with a mobile phase (gas or liquid)
& a stationary phase (column)
Chromatography – separate the molecules by adsorption, partition
and/or path (charge) differences
- Preparation and analytical chromatograpies
Resolution
1. Factors: size and rigidity of resin, packing, low
diffusion, protein concentration, selection of resin,
optimal flow rate, capacity
2. Performance: High & Low - pump & resin (pressure)
HPLC (High performance Liquid Chromatography)
3. For high resolution column chromatography
- Homogeneous small beads (homogenous packing)
- high resistance to liquid flow (high pressure
operation)
- short theoretical plate height (high resolution)
- decreasing diffusion (no line broadening, improving
resolution)
- porous bead – high capacity, size independent
experiment
(big enough for the penetration of proteins or large
molecules)
- smaller volume
General procedures of column chromatography
1.
2.
3.
4.
5.
6.
7.
(Packing the column)
Pre-equilibrium
Loading the column
Washing the column
Eluting the column
Collecting the eluting components
Detecting the eluting components
General considerations for the column chromatographic
separation
1. Sample volume, amount of protein. resolution, time,
2. Column size & packing, flow rate & chromatographic system must be
considered for maximum resolution in a short time
3. Resin (matrix)
- Condition: rigidity, nonspecific interaction, chemical stability, open
pore, small and regular size
- Cellulose, dextran, agrose, polyacrylamide or their mixture
Column chromatography
1. Chromatography on the basis of the physical property of proteins :
gel-filtration
1. Chromatography on the basis of chemical property of proteins:
ion-exchange, hydrophobic, reverse-phase, charge-transfer
2. Chromatography on the basis of the biochemcal activity of proteins :
affinity
Gel-filtration
-
-
Principle: separate proteins using the size difference
Consideration: size of protein, column (height/width of column),
sample volume, media, pore size & buffer (solubility of protein
& non-specific interaction)
Choice: Gel-filtration & desalting together, choice for the next or
previous step of ion-exchange
Elution: buffer
Gel-filtration Matrics
Applications of gel-filtration columns
1.Separation
2.Desalting: change the buffer
3. Size determination
Ion-exchange
- Principle: separate proteins using the charge difference
- Consideration: Matrix, functional residues - cation (CM, SP, S) or
anion (DEAE, QAE, Q), buffer pH & salt in buffer, PI of proteins
- Choice: usually used for the first step after initial treatment,
however high resolution ion exchange columns are also
recommended in the final stage
- Elution: salt elution or pH elution
Effect of pH on surface charge
…
Elution
…
Affinity chromatography
- Principle: separate proteins using the binding affinity difference to ligands
- Choice: best choice if you are able to make the affinity column harboring a
high selectivity to your protein (105~1011 M)
- Consideration: matrix (large surface area, less non-specific binding), ligand,
linker (spacer, steric hinderance), immobilization (cynogen bromide can be
used for attaching Lys residues to the matrix)
- General ligands: Metal binding, GST, intein, MBP
- Specific ligands: ATP, NADH, DNA, RNA, antibody & protein or chemical
ligand
- Elution: salt or specific chemicals
Charge transfer chromatography
- Metal affinity chromatography:
…
Affinity chromatography : Ligand
Affinity chromatography : activation
Hydrophobic interaction chromatography
Hydrophobic interaction column:
- Principle: separate proteins using the hydrophobicity difference
- Consideration: matrix, hydrophobicity of proteins, ligand, salt conc.
in buffer
- Choice: choice for the next step of ion exchange or AMS ppt.
- Elution : salt elution
…
Refolding
1. Principle : folding, no aggregation
2. Consideration: pH, concentration, reducing condition, additives, chaperones
3. Practice
(1) purification of the inclusion body
(2) solubilization (by chaotrophic agents such as GdHCl, urea or detergents)
(3) refolding by reducing the concentration of denaturants
one step-dialysis, step-wise dialysis, gel-filtration dilution, reverse dilution,
Mixing, solid phase refolding
(4) characterization of the refolded proteins (activity or function)
Purpose?
Antibody
production
Function
study
Structure
study
Production
Synthesis
(protein or
peptide)
Recombinant
protein
Endogenous
protein
Purity
Partially
purified
Maintenance
Short term
storage
> 95 %
Medium
term
storage
Long term
storage
Storage
Storage
1. Principle : maintaining their stability and activity
2. Factors affecting the stability of proteins: temperature ,pH, proteases,
microbial contamination, concentration, organic solvents, buffer, salt etc..
3. Practice
(1) Short term storage : keep proteins at low temperature (with protease
inhibitors)
(2) Medium term storage : keep proteins at low temperature with sodium
azide (in the case of putting them in a buffer) or in water.
(3) Long-term storage : keep proteins in deep freezer (solution or dried):
- aliquot protein solution in the amount needed for short term experiments
- do not thaw and freeze again.
Purpose?
Antibody
production
Function
study
Structure
study
Production
Synthesis
(protein or
peptide)
Recombinant
protein
Endogenous
protein
Purity
Partially
purified
Maintenance
Short term
storage
> 95 %
Medium
term
storage
Long term
storage
Why do you need proteins? (amount & purity)
1.How are you going to prepare the large amount of protein?
- from tissue or using a recombinant system.
1.How are you going to purify protein?
- using their biochemical and biophysical characters.
1.How are you going to maintain their function (activity): keep their fold
in native state.
What do you need to know?
-Protein, what is it?
-Structure & folding
-Biochemical and biophysical characters
-Function : binding & catalytic activity
-What factors affect the structure and function of protein?