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
Specific binding How to purify Proteins? Biochemists exploit the ways individual proteins differ from one another, such as solubility, size, or charge.
Column Chromatography Most common method for separating proteins
Size-exclusion chromatography Absorbance at 280 is used to identify protein-containing 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
+ + + Cl- + + + Cl- - + + + Cl- - - + + + Cl- + + + Cl- Na+ - Na+ - Na+ Na+ Na+ - - Na+ Na+ + + Na+ Cl- - Na+ Ion-Exchange chromatography - - + + Increased salt concentration
Affinity chromatography Makes use of specific binding interactions between molecules 1- Incubate crude sample with the immobilized ligand 3- Elute 2- Wash away non bound sample components from solid support
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
NTA has a tetradentate chelating group that occupies four of six sites in the nickel coordination sphere 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
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.
2+ 2+ Ni Ni Why imidazole? The imidazole ring is part of the structure of histidine
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.