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 molecules
they 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 ?
If yes, don’t need to worry about
limits *
b) A domain from a mosaic protein ?
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 there
isn’t any structural info, try secondary structure prediction
..but try several starts
and stops (primers are
cheap!)
http://www.igb.uci.edu/tools/scratch/
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 Collection
and 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 system
Advantages –
Easy, great over-expression, low
protease activity, no posttranslational modifications
Disadvantages - Protein solubility, lack of posttranslational modifications
Eukaryotic expression system
Advantages Protein solubility, post-translational
modifications
Disadvantages - Expense, low yield, proteases
Isolate protein from native source
Advantages –
Protein solubility, authenticity
Disadvantages - Expense/effort, yield, slaughter-houses
Waring blenders, Gross!(Gt)
Hierarchy – Bacteria, Yeast, SF9, Hela, native tissue
Before starting, confirm that you can make a significant
quantity 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 methods
Express 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!
electron coordination bonds
Imidazole
Nitrilotriacetic acid
pH  7.4
Kirkegaard & Perry Laboratories, Inc
If the affinity tag is removable, go back over column and collect
flow-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?
Anion exchange
(example ion exchange)
Anion #2
( Cl- )
-
Na+
+
pH=6
+
+
+
+
+
+
+
Bind (Low salt)
-
+
Na+
YFP
YFP
Anion #1
( protein )
Cl-
+
+
Na+
Cl-
+
+
+
Na+
Cl-
+
Na+
Cl-
Elute (High salt)
+
Run a 20 x (column volume) linear gradient and collect fractions
500mM NaCl
Linear gradient
(also step)
Trp, Tyr, Phe,
disulfides
50mM NaCl
example chromatogram
Run SDS-PAGE of fractions to decide which to pool
(sacrifice yield for purity?)
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.
Some proteins, usually larger proteins, can bind to
both anion and cation exchange matrices – change
pH to enhance interaction.
Electrostatic potential mapped
onto a molecular surface
Q column
SP column
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” a
bigger volume than do
large proteins
Some other chromatographic techniques
Salting out – Proteins precipitate differentially in the presence
of (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 purification
protocol. It depends on the level of purity required,
but no matter what the vendors say, there is no one
step purification to homogeneity (in my experience!)