Download - Protein purification
![Page 1: Protein purification](https://reader031.vdocuments.net/reader031/viewer/2022020320/56815f6d550346895dce75c0/html5/thumbnails/1.jpg)
Protein purification
Chromatography – Mikhail Tsvett (1901) pioneered the technique while attempting to separate yellow and red pigments from green leaves
![Page 2: Protein purification](https://reader031.vdocuments.net/reader031/viewer/2022020320/56815f6d550346895dce75c0/html5/thumbnails/2.jpg)
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.)
![Page 3: Protein purification](https://reader031.vdocuments.net/reader031/viewer/2022020320/56815f6d550346895dce75c0/html5/thumbnails/3.jpg)
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
![Page 4: Protein purification](https://reader031.vdocuments.net/reader031/viewer/2022020320/56815f6d550346895dce75c0/html5/thumbnails/4.jpg)
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!
![Page 5: Protein purification](https://reader031.vdocuments.net/reader031/viewer/2022020320/56815f6d550346895dce75c0/html5/thumbnails/5.jpg)
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!)
![Page 6: Protein purification](https://reader031.vdocuments.net/reader031/viewer/2022020320/56815f6d550346895dce75c0/html5/thumbnails/6.jpg)
Do you have the gene?
![Page 7: Protein purification](https://reader031.vdocuments.net/reader031/viewer/2022020320/56815f6d550346895dce75c0/html5/thumbnails/7.jpg)
Can look for only human
![Page 8: Protein purification](https://reader031.vdocuments.net/reader031/viewer/2022020320/56815f6d550346895dce75c0/html5/thumbnails/8.jpg)
![Page 9: Protein purification](https://reader031.vdocuments.net/reader031/viewer/2022020320/56815f6d550346895dce75c0/html5/thumbnails/9.jpg)
Lots of output!Near bottom, look for“mRNA Sequence” –
That’s the accession # you want (RefSeq record)
![Page 10: Protein purification](https://reader031.vdocuments.net/reader031/viewer/2022020320/56815f6d550346895dce75c0/html5/thumbnails/10.jpg)
Also American Type Culture Collectionand Research Genetics/Invitrogen
Integrated Molecular Analysis of Genomes and their Expression
![Page 11: Protein purification](https://reader031.vdocuments.net/reader031/viewer/2022020320/56815f6d550346895dce75c0/html5/thumbnails/11.jpg)
Shopping cart – price varies with order size
![Page 12: Protein purification](https://reader031.vdocuments.net/reader031/viewer/2022020320/56815f6d550346895dce75c0/html5/thumbnails/12.jpg)
Expression and purification of YFPBacterial 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
![Page 13: Protein purification](https://reader031.vdocuments.net/reader031/viewer/2022020320/56815f6d550346895dce75c0/html5/thumbnails/13.jpg)
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.
![Page 14: Protein purification](https://reader031.vdocuments.net/reader031/viewer/2022020320/56815f6d550346895dce75c0/html5/thumbnails/14.jpg)
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
![Page 15: Protein purification](https://reader031.vdocuments.net/reader031/viewer/2022020320/56815f6d550346895dce75c0/html5/thumbnails/15.jpg)
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?
![Page 16: Protein purification](https://reader031.vdocuments.net/reader031/viewer/2022020320/56815f6d550346895dce75c0/html5/thumbnails/16.jpg)
++
++
++
++
----
++
++
++
++
----Na+
Cl-
Cl-
Cl-
Cl-
Na+
Na+
Na+
Na+
Bind (Low salt) Elute (High salt)
Anion #1( protein )
Anion #2( Cl- )
YFP
YFP
Anion exchange(example ion exchange)
pH=6
![Page 17: Protein purification](https://reader031.vdocuments.net/reader031/viewer/2022020320/56815f6d550346895dce75c0/html5/thumbnails/17.jpg)
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)
![Page 18: Protein purification](https://reader031.vdocuments.net/reader031/viewer/2022020320/56815f6d550346895dce75c0/html5/thumbnails/18.jpg)
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
![Page 19: Protein purification](https://reader031.vdocuments.net/reader031/viewer/2022020320/56815f6d550346895dce75c0/html5/thumbnails/19.jpg)
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
![Page 20: Protein purification](https://reader031.vdocuments.net/reader031/viewer/2022020320/56815f6d550346895dce75c0/html5/thumbnails/20.jpg)
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.
![Page 21: Protein purification](https://reader031.vdocuments.net/reader031/viewer/2022020320/56815f6d550346895dce75c0/html5/thumbnails/21.jpg)
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!)