
His
6
Tagged
Based on Ni chelation by poly-histidine (His
6
), 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”

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.

FT/Wash
Affinity Column
Elution
Elution
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.
Protease

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.

•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

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.

• 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
Isolation of His
6
-Signaling Domain of Tar chemoreceptor in Inclusion bodies
Samples were separated by 15% SDS-
PAGE. Note that the first three samples are over-loaded.
His
6
-SD
Lysate 1, ‘low speed’ supernatant
Lysate 2, ‘high speed’ supernatant
Pellet 2, ‘high speed’ pellet
Pellet 1, ‘low speed’ pellet
= Inclusion bodies

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

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.

PMSF (Phenylmethyl-sulfonylfluoride) Inhibits serine proteases.
Also inhibits cysteine
EDTA, EGTA
Pepstatin
Leupeptin
Cysteine
Inhibit metalloproteinases
Inhibits Aspartic Proteases
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

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.

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 ( 'saltingin’ ). 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 ( 'saltingout’ ).
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.

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
---
2
2
DEAE-resin (diethylaminoethyl-) positively charged , i.e. anion exchanger
2
3
2
2
2
3

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.

Large molecules do not penetrate the pores of the support, and elute in the void volume (V o
); medium sized molecules penetrate the support to some degree and elute in a volume (V e
) between V o and V t
; the small molecules are so small that they penetrate the pore volume (V p
) of the support completely and elute in the total volume (V t
).

Bio-Rad Bio-Silect SEC 125-5 column
Thyroglobulin 670,000
IgG 150,000
Ovalbumin
Myoglobin
43,000
17,000
Vitamin B12 1,350

Matrix name Bead type Approximate fractionation range peptides and globular proteins
(molecular weight)
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
¹Sephadex is a registered trademark of Pharmacia PL.
³Bio Gel is a registered trademark of Bio-Rad Laboratories, Inc.
1500 - 30000
4000 - 150000
5000 - 250000
3000 - 60000
15000 - 150000
60000 - 400000

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 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 .

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)