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Protein Purification and

Crystallization

Outline

Bacterial
Transformati
on

Induction
and Protein
Extraction

Protein
Purification

Protein
Crystallizati
on

Bacterial Transformation
Two MCS for
expression of
two proteins in
same bacterial
cell
simultaneously

Production of lacI
repressor protein so
that protein of
interest not

Lac operator
for regulation
of transcription
of gene of
interest

Replication of
plasmid in
bacteria

Strong
promoter for
upregulated
expression of
proteins of
interest in
bacteria

For subsequent
protein
purification

Induction
- IPTG (lactose analogue) binds to and inactivates lacI repressor protein
no longer bound to lac operator
- RNA polymerase can bind to T7 promoter for transcription of gene of
interest
- Incubate at lower than room temperature for proper protein folding
and to minimize precipitation (loss of yield)

Extraction
- Lysozyme to break down peptidoglycan cell wall
- PMSF to inhibit proteases
- DNase I to break down sticky DNA
- Sonication to lyse bacterial cells and release proteins
- Centrifugation: Cell debris in pellet, proteins in supernatant

Protein Purification

Affinity Chromatography

Resin beads bound


with
Ni2+ or Co2+ for
His tagged
proteins
OR
Glutathione
(cysteine, glycine,
glutamine) for
glutathione Stransferase
tagged proteins

Equilibrate column with buffer so that protein of interest is not


denatured

Add supernatant to column: His tagged protein of interest binds to


resin beads in column while most other proteins run out of column

Wash column with buffer to remove non-specific binding

Elute protein of interest from column using competitive ligand (e.g.


imidazole for His tagged proteins or reduced glutathione for GST
tagged proteins) or by changing the pH, ionic strength or polarity of
the solvent (but may risk denaturing protein)

Application: Antibody Purification


High affinity and specificity of
Protein A and protein G for the fc-region (fragment
crystallisable region, i.e. tail part) of IgG
Protein L for the kappa light chains of antibodies.

Antibody is often eluted by lowering the pH.

Desalting
Replacing the existing buffer solution containing
imidazole or reduced glutathione with fresh buffer
without these substances
Why?
After specific protease cleavage to cleave the His tag or GST
tag from the protein of interest, the mixture has to be run
through a second affinity column.
The protein of interest is collected in the flow through and
wash.
The tags are bound to the column.
If the imidazole or reduced glutathione is not removed from
the buffer, the tags will fail to bind to the column and cannot

Ion Exchange Chromatography


Net surface charge of protein varies according to surrounding
pH
Above pI, the net surface charge would be negative protein
would bind to positively charged cations bound to column
Below pI, the net surface charge would be positive protein
would bind to negatively charged anions bound to column
Proteins bind as they are loaded onto a column at low ionic
strength.
Gradient elution: Increasing salt concentration or changing pH.

Hydrophobic Interaction
Chromatography
Hydrophobic proteins bind to the hydrophobic surface of
the HIC column as they are loaded at high ionic
strength.
As the ionic strength of the buffer is reduced, the
protein with the lowest degree of hydrophobicity is
eluted first.
The stationary phase for reversed phase HPLC is more
hydrophobic elution must be done with non-polar
organic solvents which has a higher risk of denaturing
proteins.

Size Exclusion Chromatography (Gel


Filtration)
Beads in Sephadex (cross-linked dextran) column lack
reactive and adsorptive properties.
Molecules larger than the pores are unable to diffuse
into the beads elute first.
Molecules smaller than the pores can penetrate the
pores (enters the total pore volume) to varying degrees
based on their size. The smaller the molecule, the later
it is eluted.

Checking Purity with SDS PAGE


Sodium dodecyl sulfate (SDS) denatures proteins into polypeptides and
confers upon them a net negative charge (1 SDS: 2 aa)
Heating with strong reducing agent to reduce disulfide linkages
Negatively charged polypeptides migrate towards anode. Greater Mr,
slower migration rate.
Linear relationship between log(Mr) and migration distance (Rf)
Higher bisacrylamide: acrylamide ratio and higher acrylamide conc.,
smaller pore size, better resolution.
How is Native PAGE different?
Proteins are not denatured.
Shape has more impact on migration rate than Mr.
Long asymmetric molecules migrate slower than spherical molecules.

Quantification of yield
Nanodrop = mini spectrophotometer
Wipe pedestal with deionized water and Kimwipe (!)
Load blank, press blank
Load sample, press measure
Wipe pedestal with deionized water and Kimwipe to
prevent contamination (!)

Protein Crystallization

Factors
Sample purity and homogeneity
Formation of a well-ordered crystal lattice, which promotes protein crystallization.

Temperature
As temperature increases, proteins whose crystallization is enthalpy-driven become more
soluble while proteins whose crystallization is entropy-driven become less soluble.

Ionic strength
The change in protein solubility with temperature is greater at low ionic strength than at
high ionic strength.

pH
R groups of the amino acid residues in the protein have different ionization states, affecting
the formation of ionic bonds and hydrogen bonds important for the formation of specific
crystal contacts.

Precipitant type and concentration


Higher concentration, lower solubility.

Crystallization method
Sitting-drop vapor diffusion
method

Hanging-drop vapor
diffusion method

Initial reagent concentration in the droplet < in the reservoir solution.

Over time, water vapour will diffuse from the droplet to the reservoir solution.

The protein sample is thus concentrated, increasing the relative supersaturation of the protein sample in the drop.

Protein Solubility Curve