Molecular)Cell)Biologymcb5068.wustl.edu/MCB/Lecturers/Cooper/s/5_ProteinPurification.pdf ·...

Molecular Cell Biology

Purifying Proteins

Cooper

Wednesday, August 29, 2012

References

• Nelson and Cox, Lehninger’s Principles of Biochemistry, 3rd edi@on, pp. 130-‐137.

• Wikipedia: en.wikipedia.org/wiki/Protein_purifica@on

• Lecture Presenta@on by Dr. Chrisopher Fraser: mcb.berkeley.edu/courses/mcb102/


Why Purify Proteins?

• Protein research: Understand func@on and structure of individual proteins

• Drugs targe@ng proteins– Insulin

– Growth Hormone

– Erythropoie@n (EPO)

– Interferon

– Hercep@n


Step 1: Assay for the Protein of Interest

• Enzymes: Reac@ons they Catalyse

• Biological effects: Hormones

• Immunochemistry: An@bodies that Recognize the Protein


• Preserve the structure during purification• Consider that the structure may be lost• Activity assay a good test

Levels of Protein Structure

• Preserve the Structure during Purifica@on

• Consider that the Structure May Be Lost

• Ac@vity Assay is a Good Test


Star@ng the Purifica@on

• Biological Source: Need @ssue or cell type that contains the protein, in large amounts

• Lyse Cells: Homogenize / Disrupt

• Separate Cell Homogenate into Frac@ons


Prevent Proteolysisand Keep the Protein Stable

• Cold temperature– Also inhibits growth of bacteria

• Chemicals that Inhibit Proteases– EDTA to inhibit Ca++ dependent proteases

• Test a variety of buffer condi@ons– Find one where enzyme ac@vity remains constant over @me


Purifica@on Techniques

• Based on Various Chemical and Physical Proper@es of Proteins

– Solubility (func@on of salt, pH, temperature)

– Charge

– Size

– Binding proper@es (specific ligands)


Ammonium Sulfate Precipita@on

• Very high ionic strength -‐ Proteins precipitate -‐ “Sal@ng Out”

• Modest Purifica@on but Also Useful to Concentrate the Sample


Dialysis to Change Solu@on Condi@ons

• For Various Steps, ogen want protein mixture to be in a certain buffer

• Dialyse the sample against a semi-‐permeable membrane


Column Chromatography: General Principles


Ion-‐Exchange Chromatography


Size-‐Exclusion (Gel Filtra@on) Chromatography


Affinity Chromatography


Purifica@on Table: Specific Ac@vity

Specific Ac@vity = Ac@vity / Total Protein


SDS PolyAcrylamide Gel Electrophoresis


Using SDS-‐PAGE to Assess Purity

Separate the Polypep@des

MigrateBased on Mass


Determining Mass of Polypep@de


Isoelectric Focusing Electrophoresis(Posi@on Depends on pI of Protein)


Isoelectric Points of Some Proteins


Two-‐Dimensional Electrophoresis

First Dimension: Isoelectric Focusing

Second Dimension: SDS-‐PAGE

IEF Tube Gel Placed on Top of SDS poly-‐

acrylamide gel


Typical 2-D Gel from Whole Cell or Tissue Sample


Affinity-‐tag Purifica@on

• Use Recombinant DNA Technology to Create a Fusion Protein containing the Tag and Your Favorite Protein (YFP).


Affinity-‐tag Purifica@on

• Op@onal: Place a very specific (i.e. rate) proteoly@c cleavage site between the Tag and YFP.– TEV Protease: ENLYFQG

– PreScission Protease: LEVLFQ/GP

• Express in bacteria, yeast, insect or mammalian cells


Advantages and Disadvantages of Protein Expression Systems

• www.genwaybio.com/gw_file.php?fid=6033

• Bacteria: Large amounts of protein, Easy to grow. No post-‐transla@onal modifica@ons. Low cost.

• Yeast: Intermediate Ease of Growth and Yield of Protein. Some modifica@ons: Lipid, Carbohydrate. Low cost.

• Insect Cells: Intermediate Ease of Growth and Yield of Protein. Baculovirus. High cost.

• Mammalian Cells: Full range of modifica@ons. Poor yield, hard to grow. High cost.


Example of a GST Expression Plasmid for Yeast

Your ProteinGoesHere


Affinity Tags Used for Purifica@on of Recombinant Proteins

• GST: Glutathione-‐S-‐Transferase. Purify w/ column of glutathione. Elute w/ same.

• MBP: Maltose-‐Binding Protein. Purify w/ column of amylose. Elute w/ maltose.

• 6xHis: Poly-‐His@dine. Purify w/ metal (Nickel or Cobalt) resins. Elute w/ imidazole or low pH.


An@body-‐based Purifica@on

• Fuse a short Pep@de to Your Favorite Protein– HA: YPYDVPDYA

– Myc: EQKLISEEDL

– FLAG: DYKDDDDK

• Purify w/ affinity beads containing a monoclonal an@body that binds @ghtly to the pep@de

• Elute w/ pep@de


TAP: Tandem Affinity Purifica@on

• Two tags -‐ Protein A and Calmodulin-‐binding pep@de, separated by a proteoly@c cleavage site.– Purify on IgG beads first.

– Cleave with highly specific protease (TEV) to elute

– Purify on Calmodulin beads. Elute w/ EGTA.


Concentra@ng the Purified Protein

• Ogen Need to Increase Protein Concentra@on at End of Protocol

• Dialyze into water +/-‐ sucrose. Lyophilize (freeze-‐dry).

• Ultrafiltra@on. Force through a semi-‐permeable membrane.

• Ion-‐exchange chromatography column w/ large step increase in salt.


Determining Protein Concentra@on

• Absorbance (ultraviolet): 280 nm. Depends on number of Trp, Tyr and Phe residues in the protein.– Calculate / Predict ex@nc@on coefficient

– Lower wavelength (230 or 205 nm) alterna@ve

• Colored Dye Absorbance– Lowry, Biuret, Bradford Assays

– Absorb at Higher Wavelength (e.g. 595 nm)


End


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