Transcript PowerPoint

Examples of Fusion Systems for Protein Purification:
 His6 Tagged
Based on Ni chelation by poly-histidine (His6), Several vectors and
fusion types available (several companies), Versatile system, many
“accessories”, refolding inclusion bodies, Works well with only a small
addition to N- or C-terminus or with larger “protease sensitive linker”
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Maltose Binding Protein Fusions
GST fusions
Flag-Tag Fusions
Inteins Fusions
These three systems generate large protein fusions and are
purified based on the properties of the fused protein. All work well but the
addition of the fusion can be a problem sometimes. Inteins are “self
cleaving” in DTT , if your protein can tolerate high concentrations of
reducing agent.
General Principle of Use of Fusion Proteins for Purification
FT/Wash
Affinity Column
Elution
Elution
Protease
FT/Wash
The fusion protein is NOT the native proteins
Can impair function. Typically not good for structural studies.
The Proteases are specific and expensive
A problem when needing large amounts of proteins.
Maltose Binding Protein: malE gene fusions
pMAL-c2 The malE gene on this vector has a deletion of the signal sequence,
leading to cytoplasmic expression of the fusion protein.
pMAL-p2 The signal sequence of the malE gene on this vector is intact,
potentially allowing fusion proteins to be exported to the periplasm.
•if it is known that the protein of interest does not fold properly in the E. coli
cytoplasm,
•if it requires disulfide bonding to fold correctly. pMAL ™ -p2 may also be the
•if the protein of interest is a secreted protein, or extracellular domain of a
transmembrane protein.
Grow cells
Sample 1: uninduced cells
Add IPTG
Sample 2: induced cells
Divide culture and harvest cells
For pMAL-c2 and -p2constructs
For pMAL-p2 constructs only
Resuspend in column buffer
Prepare cytoplasmic extract
Sample 3: crude extract
Sample 4: insoluble matter
Resuspend in Tris/sucrose
Prepare periplasmic extract
Sample 6: periplasmic extract
(osmotic-shock fluid)
Test amylose resin binding
Sample 5: protein bound to amylose
SDS-PAGE
SDS-polyacrylamide gel electrophoresis of fractions from
the purification of MBP-paramyosin-DSal.
A: Lane 1: uninduced cells. Lane 2: induced cells.
B: Lane 1: purified protein eluted from amylose column with maltose. Lane 2: purified
protein after Factor Xa cleavage. Lane 3: paramyosin fragment eluted from second
amylose column.
Optimizing Expression of the Protein
•Temperature for growing cultures
•Media (rich vs poor)
•Does the protein require specific cofactors
These can become limiting when over expressing
•When is the protein made
exponential / stationary phase, aerobic / anaerobic, catabolite repressed
•Is the protein more stable or less stable under certain conditions
exponential / stationary phase
•Is the protein stable to freezing or storage
Most proteins tolerate freezing under appropriate conditions (such as 10% glycerol)
but do not endure repeated freeze/thaw cycles
For inducible expression systems:
•How much inducer is optimal
•When to induce and for how long
•Which is the best expression system for the protein of interest
Over-expressed proteins and Solubility
Often an over-expressed protein will have limited solubility in the cell. These
leads to the formation of inclusion bodies.
Minimizing Inclusion Body Formation
•Temperature
•Media
•Level of Induction
In general reducing expression levels will help reduce inclusion body
formation. This is often most easily accomplished by adjusting temperature and
media. These inducible expression systems are difficult to regulate with inducer
levels.
•Strain Background
•Co-induction of chaperonins
Some success has been had by using strains that overproduce chaperonins but
these have not been generally applicable. Inclusion body formation likely leads
to induction of the pathways anyway.
Inclusion bodies are almost PURE protein!
• This can be good if function is not required (e.g. to raise antibodies)
• Often conditions can be found for refolding of the denatured protein
Pellet 1
Pellet 2
Lystae 2
Lystae 1
Cells
Markers
Isolation of His6-Signaling Domain of Tar chemoreceptor in Inclusion bodies
Samples were separated by 15% SDSPAGE. Note that the first three samples are
over-loaded.
His6-SD
Lysate 1, ‘low speed’ supernatant
Lysate 2, ‘high speed’ supernatant
Pellet 2, ‘high speed’ pellet
Pellet 1, ‘low speed’ pellet
= Inclusion bodies
Reducing agents
The bacterial cytoplasm is reducing so there are no disulfide bonds (oxidized
state) in cytoplasmic proteins, therefore if you have cysteine residues in your
protein you should include reducing agents. Some level of reducing agents should
be added for periplasmic proteins as well. If you have no cysteine residues in
your protein, then this is not so important.
DTT (dithiothreitol)
b-mercaptoethanol
Proteases
Bacterial proteases are acting against you during a purification. Always
follow the following general rules:
•work fast especially during the early steps until most of the proteases are separated from
your protein. (many of the cells most active non-specific proteases are in the periplasm and
problems do not start until you lyse the cells.
•Use Protease inhibitors- as a general rule have EDTA in all your buffers unless it is a
problem for your specific protein (if your protein requires Mg for activity be sure to account
for the EDTA in an assays). PMSF is very effective but used usually only during the early
steps.
•keep everything cold- prechill rotors, centrifuges, bottles, buffers. Always keep your
fractions on ice. When sonicating, do it in short bursts with intervals to cool the sonication
tip and sample in between.
Commonly Used Protease Inhibitors
PMSF (Phenylmethyl-sulfonylfluoride)
Also
Inhibits serine proteases.
inhibits cysteine
EDTA, EGTA
Inhibit metalloproteinases
Pepstatin
Inhibits Aspartic Proteases
Leupeptin
Cysteine
Inhibits Serine and
Proteases
Protease Cocktail Mixes: Mixture of protease inhibitors in one
complete tablet can stop a multitude of proteases including serine
proteases, cysteine proteases and metalloproteases.
http://biochem.boehringer-mannheim.com/prodinfo_fst.htm?/prod_inf/manuals/protease/prot_toc.htm
Lysis of Bacterial Cells
Bacteria are typically quite resistant to lysis and special methods are
required for breaking open the cells. Gram positive cells tend to be more
troublesome than gram negative cells.
Ultra-sonication
French Press
Enzymatic digestion of cell wall
Repeated Freeze-thaw cycles
Precipitation Steps
The solubility of proteins varies according to the ionic strength and
hence according to the salt concentration of the solution. At low
concentrations of salt, the solubility of the protein increases with salt
concentration ('salting-in’). However, as the salt concentration is
increased still further, the solubility of the protein begins to decrease. At
sufficiently high ionic strength the protein solubility will have decreased
to the point where the protein will be almost completely precipitated
from solution ( 'salting-out’).
In practice, ammonium sulfate is the salt commonly used since
it is highly water-soluble, relatively cheap and available at high purity.
Furthermore it has no adverse effects upon enzyme activity.
Concentrators - previously salting out was used to concentrate proteins in
addition to fractionation. Concentration of proteins is now often done using
commercially available ultra-filtration devices that concentrate proteins
quickly and efficiently.
Ion-exchange chromatography
An ion-exchange resin consists of an insoluble matrix with charge groups covalently
attached. Negatively charged exchangers bind positively charged ions (cations) They can
bind one type of cation but, when presented with a second type of cation, this may
displace, or exchange with, the first. Hence these resins are called cation-exchange
resins. Similarly anion-exchange resins are positively charged and bind (and exchange)
negatively charged ions (anions).
A Cation exchange resin with bound positive counterions
B Anion exchange resin with bound negative counterions
Proteins are charged molecules. The overall number of charges on a particular
protein at a particular pH will depend on the number and type of ionizable amino
acid side chain groups it contains. For any one protein there will be a pH at which
the overall number of negative charges equals the number of positive charges and so
it has no net charge. This is its isoelectric point (pI). At this pH the protein will not
bind to any ion-exchange resin. Below this pH the protein will have a net positive
charge and will bind to a cation exchanger, whilst above this pH it will have a net
negative charge and bind to an anion exchanger.
CM-resin (carboxymethyl-) negatively charged, i.e. cation exchanger
---CH2OCH2COO¯
DEAE-resin (diethylaminoethyl-) positively charged , i.e. anion exchanger
CH2CH3
---CH2CH2N +
CH2CH3
Gel Filtration Chromatography
A wide range of biological molecules can be separated on the basis of
differences in their size and shape which lead to differences in their ability
to penetrate porous matrices. This procedure is also known as molecular
sieve chromatography or molecular exclusion chromatography.
It is important to note that shape is a very important characteristic
in gel filtration chromatography. Determination of Molecular weight by this
method is fraught with difficulties and complications. Combined with other
methods, such as light scattering, can be very powerful for determination of
molecular weight of proteins and complexes.
Two proteins of very different size maybe behave similarly on Gel Filtration
column if they have similar ‘radius of gyration”. Also multi-domain proteins
connected by flexible linkers can behave quite atypical.
Mechanism of size exclusion chromatography
Large molecules do not penetrate the pores of the support, and elute in the
void volume (Vo); medium sized molecules penetrate the support to some
degree and elute in a volume (Ve) between Vo and Vt; the small molecules
are so small that they penetrate the pore volume (Vp) of the support
completely and elute in the total volume (Vt).
Typical Gel Filtration Fractionation
Bio-Rad Bio-Silect SEC 125-5 column
Retention Time
Thyroglobulin
670,000
IgG
150,000
Ovalbumin
43,000
Myoglobin
17,000
Vitamin B12
1,350
Commonly Used Gel Filtration Media
Matrix name
Bead type
Sephadex G-50¹
Sephadex G-100¹
Sephacryl S-200 HR¹
Bio-Gel P-60³
Bio Gel P-150³
Bio-Gel P-300³
dextran
dextran
dextran
polyacrylamide
polyacrylamide
polyacrylamide
Approximate fractionation range
peptides and globular proteins
(molecular weight)
1500 - 30000
4000 - 150000
5000 - 250000
3000 - 60000
15000 - 150000
60000 - 400000
¹Sephadex is a registered trademark of Pharmacia PL.
³Bio Gel is a registered trademark of Bio-Rad Laboratories, Inc.
Hydrophobic Interaction Chromatography
Hydrophobic Interaction Chromatography (HIC) separates
proteins with different hydrophobicities based on the reversible
interaction of a protein and a hydrophobic surface.
Even soluble proteins will have a significant amount of
hydrophobic character (as much as 70% of the amino acid residues on the
surface of a protein may be hydrophobic).
High ionic strength stabilizes hydrophobic interactions, therefore
the samples are loaded in high salt and bound proteins are eluted with
decreasing salt concentrations. Ammonium sulfate is often used as the
salt so this is an ideal chromatography method to follow an ammonium
sulfate precipitation step.
Affinity Chromatography
Affinity Chromatography separates on the basis of a reversible
interaction between the protein(s) and a specific ligand attached to a
resin. Typically is highly selective and therefore has high resolution and
usually high capacity.
The purification of specific fusion proteins (e.g. His- tagged,
MBP, GST fusions) are all examples of affinity chromatography. A
column generated using antibodies specific for the protein of interest is
another example, often referred to as Immuno-affinity chromatography.
A note about instrumentation
1) Standard chromatography
Gravity fed or peristaltic pump, typically done in cold room, easy
effective and inexpensive
2) FPLC (fast protein liquid chromatography)
medium pressure and flow rates, can be set up in cold room, expensive
but really just a toned down version of an HPLC
2) HPLC (high performance liquid chromatography)
high pressure and flow rates, usually run at room temp, relatively
expensive but VERY reproducible and reliable (if carefully maintained)