Protein Manipulation

김경규( 성균관대 의학과 )

Overview of Biochemical Experiments (characterization)

(Partially) Purified

biological materials

Physicochemical characterization:MW (electrophoresis=EP, mass), size (EP, gel-filtration, centrifuge), purity (EP, chromatography), concentration (Bradford assay, spectroscopy), PI: electrofocusing

Functional assayIdentification (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 study2nd structure (CD, IR), 3rd structure (EM, X-ray, NMR), distance (fluorescence), conformational change (fluorescence, CD, UV-VIS, IR)

ImmunochemistryAntibody production

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

productionFunction

studyStructure

study

ProductionSynthesis(protein or peptide)

Recombinant protein

Endogenous protein

Purity

Maintenance

Partially purified

> 95 %

Short term storage

Medium term

storage

Long term storage

Antibody production

Protein Chemistry

Protein → Amino Acids

20 Amino Acids

Protein folding

Alanine 7.2% Methionine 2.2%Cysteine 1.9% Asparagine 4.3%Aspartic acid 5.3% Proline 5.2%Glutamic acid 6.3% Glutamine 4.3%Phenylalanine 3.9% Arginine 5.1%Glycine 7.2% Serine 6.8%Histidine 2.3% Threonine 5.9%Isoleucine 5.3% Valine 6.6%Lysine 5.9% Tryptophane 1.4%Leucine 9.1% 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

productionFunction

studyStructure

study

ProductionSynthesis(protein or peptide)

Recombinant protein

Endogenous protein

Purity

Maintenance

Partially purified

> 95 %

Short term storage

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

MarkerOri

- 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)

Prokaryotic expression:

low expression level or insoluble protein

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 - proceduresPrimer design PCR Purification of PCR productRestriction enzyme treatmentGel extraction of the restricted PCR productLigation of PCR product with digested vectorTransformation into BL21(DE3)Colony selectionCulture & IPTG inductionCell harvestExpression test in SDS-PAGEPurification

lacI

KanR

F1 origin

pVFT1L vector5378bp

XhoI

NotI

HindIII

SalI

EcoRI

BamHI

NcoI

TEV site

KpnI

NheI

NdeI

NcoI

lacI

KanR

F1 origin

pVFT1L vector5378bp

XhoI

NotI

HindIII

SalI

EcoRI

BamHI

NcoI

TEV site

KpnI

NheI

NdeI

NcoI

Insect cell expressionSf9 cells

Sf9 cells transfected with b-gal

Transfection

Expressed proteins

Plaques

Virus purification

infection

(Modified from Invitrogen Catalogue)

Mammalian cell expression system : CHO cells

No NTPs

DHFR+YGI+ Grow cells and

collect conditioned serum free media

DHFR

CHO

DHFR-

5 ug100 ug

YGI

100-500X DHFR

Add MTX

(DHFR inhibitor)

100-500X YGI

YPI

YPI

YPI

YPI YPI

YPI YPI

Mammalian cell expression system: 293 or Hella cells

Selection

By marker

YGI+

Grow cells and collect conditioned serum free media

293 cells

YGIVirus infectionor transfection

Colony selection

YPI

YPI

YPI

YPI YPI

YPI YPI

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

productionFunction

studyStructure

study

ProductionSynthesis(protein or peptide)

Recombinant protein

Endogenous protein

Purity

Maintenance

Partially purified

> 95 %

Short term storage

Medium term

storage

Long term storage

Protein purification

References :Protein purification , Robert Scopes, Springer-VerlagModern experimental biochemistry, Rodney Boyer, Benjamin CummingsProtein purification techniques, Simon Roe, OxfordGE healthcare catalogue, . 

General consideration for purification

1. Maximize the yield and minimize the cost and time2. Separate proteins using their physicochemical

characters3. Remove major impurity first4. Use the most effective method first5. Use the most expensive method last6. Make your protein active

Protein handling:

1. Principle: keep protein active and stable2. Factors: contamination, denaturation, preteolytic

digestion, chemical modification, adsorption3. 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

Cell Paste

Crude extract

Partially purified (1st) protein

Partially purified (2nd) protein

Purified protein

Function studies

Cell disruption and debris separation

Protein overexpression or raw materials

Pretreatment of samples or low-resolution chromatography

Protein separation using chromatography

Protein separation using high-resolution chromatography

Purity < 95 %

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. (Packing the column)2. Pre-equilibrium3. Loading the column4. Washing the column5. Eluting the column6. Collecting the eluting components7. 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 time3. 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-filtration1. Chromatography on the basis of chemical property of proteins:

ion-exchange, hydrophobic, reverse-phase, charge-transfer2. 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.Separation2.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 aggregation2. Consideration: pH, concentration, reducing condition, additives, chaperones3. 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

productionFunction

studyStructure

study

ProductionSynthesis(protein or peptide)

Recombinant protein

Endogenous protein

Purity

Maintenance

Partially purified

> 95 %

Short term storage

Medium term

storage

Long term storage

Storage

Storage

1. Principle : maintaining their stability and activity2. 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

productionFunction

studyStructure

study

ProductionSynthesis(protein or peptide)

Recombinant protein

Endogenous protein

Purity

Maintenance

Partially purified

> 95 %

Short term storage

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?