Protein purification

Chromatography – Mikhail Tsvett (1901) pioneered the technique while attempting to separate yellow and red pigments from green leaves

If you don’t have the gene that encodes the protein but you have a source, you may want to purify the protein for any of the following reasons:

The purified protein can be used to determine the amino acid sequence.

The purified protein can be used to make antibodies.

The purified protein can be analyzed and identified by mass spectroscopy.

If you have the gene that encodes the protein, you may want to purify the protein for other reasons:

Pure proteins are required for structural analysis.(x-ray crystallography and NMR spectroscopy).

Pure proteins are required to study enzyme function.

Pure proteins can be used to determine what other proteins or moleculesthey might interact with.

Pure proteins are needed for studies of protein function (e.g. Are there regulatory subunits? Is it phosphorylated? Is the protein regulated by its interactions with other proteins? Etc.)

So you want to make a pure protein?

a) An entire protein ?

b) A domain from a mosaic protein ?

If yes, don’t need to worry about limits *

Need to worry about limits

Learn all you can before beginning

MSA can often give you ideas for deciding on construct limits

Even better if there’s some structural information!

If multiple sequence alignments do not help and thereisn’t any structural info, try secondary structure prediction

http://www.igb.uci.edu/tools/scratch/

..but try several startsand stops (primers arecheap!)

Do you have the gene?

Can look for only human

Lots of output!Near bottom, look for“mRNA Sequence” –

That’s the accession # you want (RefSeq record)

Also American Type Culture Collectionand Research Genetics/Invitrogen

Integrated Molecular Analysis of Genomes and their Expression

Shopping cart – price varies with order size

Expression and purification of YFP

Bacterial expression systemAdvantages – Easy, great over-expression, low

protease activity, no post-translational modifications

Disadvantages - Protein solubility, lack of post- translational modifications

Eukaryotic expression systemAdvantages - Protein solubility, post-translational

modificationsDisadvantages - Expense, low yield, proteases

Isolate protein from native sourceAdvantages – Protein solubility, authenticityDisadvantages - Expense/effort, yield, slaughter-houses

Waring blenders, Gross!(Gt)

Hierarchy – Bacteria, Yeast, SF9, Hela, native tissue

Before starting, confirm that you can make a significantquantity of soluble protein. Small scale solubility experiments are very important and typically will involve varying inducer

concentration, expression temperature, expression construct,etc.

Each protein is unique – must exploit differences

Particular affinities GST, 6xHis, antibodies

Solubility (NH4)2SO4, PEG precip.

Charge ion exchange

Hydrophobicity hydrophobic chromatography

Size gel exclusion

Iso-electric point iso-electric focusing

Thermal stability alter temp.

Standard methodsExpress protein in frame with an affinity tag – often tag is removable with a protease.Common tags: 6xHis, GST, CaM, MBP. Use affinity chromatography for first step!

Nitrilotriacetic acid

Imidazole

pH 7.4

electron coordination bonds

Kirkegaard & Perry Laboratories, Inc

If the affinity tag is removable, go back over column and collectflow-through (or digest on the column).

Ion exchange chromatography (what is the theoretical pI of your protein?)DiEthylAminoEthane (DEAE), CarboxyMethyl (CM), Quaternary amine, Sulfonic acid.

http://www.proteinchemist.com/tutorial/iec.html

These functional groups are charged over a broad pH range. Why would that be desirable?

+

++

++

+

+

+

----

+

++

++

+

+

+

----Na+

Cl-

Cl-

Cl-

Cl-

Na+

Na+

Na+

Na+

Bind (Low salt) Elute (High salt)

Anion #1( protein )

Anion #2( Cl- )

YF

P

YF

P

Anion exchange(example ion exchange)

pH=6

50mM NaCl

500mM NaCl

Run a 20 x (column volume) linear gradient and collect fractions

example chromatogram

Run SDS-PAGE of fractions to decide which to pool(sacrifice yield for purity?)

Trp, Tyr, Phe,disulfides

Linear gradient(also step)

Some proteins, usually larger proteins, can bind toboth anion and cation exchange matrices – change

pH to enhance interaction.

Stronger and higher resolution ion exchange media (Q, SP) may be employed to separate proteins that were not baseline

separated with weak ion exchange step.

Q column SP column

Electrostatic potential mappedonto a molecular surface

Gel exclusion chromatography – Separates proteins by size. Your protein should elute at the proper volume for its expected Mr.

Want a nice, symmetric peak in the chromatogram.

Small proteins “see” abigger volume than do large proteins

Some other chromatographic techniques

Salting out – Proteins precipitate differentially in the presenceof (NH4)2SO4 or polyethylene glycol - It’s probably worth trying

Hydrophobic – Load proteins onto phenyl sepharose in presence of ~1.5M (NH4)2SO4 and run decreasing [(NH4)2SO4]gradient. More hydrophobic elutes later.

Isoelectric focusing – Electrophorese protein in matrix containing pH gradient. When the protein reaches that pH where it has no net charge it ceases to migrate. Retrieve protein from matrix.

What one does in practice is discover a purificationprotocol. It depends on the level of purity required,but no matter what the vendors say, there is no onestep purification to homogeneity (in my experience!)