Transcript Protein Purification - Home for HASPI, San Diego's Health

Protein Purification
Why purify Proteins?
 Characterize
 Function
 Activity
 Structure
 Study protein regulation and protein interactions
 Use in assays
 Produce Antibodies
 Perform structural analysis by X-Ray and
Crystallography
Where do you get proteins?
 Nature Proteins (from organism)
 Protein expression systems
 Genes cloned into a vector, inserted into a living organism
which makes the protein
 Two types
 Constitutive production
 Inducible
How to purify Proteins?
Biochemists exploit the ways individual proteins differ
from one another, such as solubility, size, or charge.
Specific
binding
Column Chromatography
Most common method for separating proteins
Size-exclusion
chromatography
Size-exclusion
chromatography
Absorbance at 280 is used to identify proteincontaining fractions. You can also perform an
enzyme specific assay.
Ion-Exchange chromatography
-
-
+
+
(-)
(-)
(+)
(+)
If pH mobile phase =7.2
Then charge of the proteins:
++
++
+
++
+
-
-
-
-
+
+
++
+
++
Anion exchange column = + charged
+
+
+
Ion-Exchange chromatography
-
+
-
+
+
-
-
+
+
+
+
+
Cl- +
+
Cl-
+
Cl-
+
+
+ Cl-
+
+
Na+ Na+ -
+ Cl-
Na+
Na+ -
Na+Na+
+
Increased salt concentration
Na+
Na+ Na+
Cl-
+
Affinity chromatography
Makes use of specific binding interactions between molecules
1- Incubate crude sample with
the immobilized ligand
2- Wash away non bound sample
components from solid support
3- Elute
Affinity chromatography
 Commonly used affinity columns:




Ni2+  binds to poly Histidines (example 6xHis)
Specific antibodies (anti-Flag tag)
glutathione  binds to GST
Protein A or G  binds antibodies
Affinity chromatography
 Possible elution strategies:




pH
Ion strengh
Denature
Competitor ligand or analog
Ni-NTA columns
The high affinity of the Ni-NTA resins for 6xHis-tagged
proteins or peptides is due to:
1- the strength with which these ions are held to the NTA resin
NTA has a tetradentate chelating
group that occupies four of six sites
in the nickel coordination sphere
2- the specificity of the interaction between histidine
residues and immobilized nickel ions
Protein elution
Elution of His tagged proteins can be achieved either by
reducing the pH, or by competition with imidazole.
 Monomers are generally eluted at approximately pH 5.9
or with imidazole concentrations greater than 100 mM,
 Multimers elute at around pH 4.5 or 200 mM imidazole.
Why imidazole?
The imidazole ring is part
of the
structure of histidine
Ni
2+
Ni
2+
Protein yield
Using a Ni-NTA spin column up to 150 µg of 6xHis-tagged
protein can be purified to up to 90% homogeneity.
Actual yields and purity will vary depending on the size
and expression level of the recombinant protein, as well
as the viscosity of the lysate.
Specific considerations
Contaminating proteins
Proteins that contain neighboring histidines are not
common in bacteria, but are quite abundant in eukaryotic
cells.
In some rare cases non-tagged, cellular proteins may bind
to the Ni-NTA.