Protein Purification Lecture

1. Protein Purification
What is the table below and where have you seen it before?
Shafqat et al. Eur. J. Biochem. 263, 305-311 (1999)

2. Overall Strategy
Molecular characteristics of target protein
Look for the properties that
distinguish the target
from other proteins
Target features
charge/hydrophobicity
solubility & stabilization
size
Define a way of identifying the presence of your
protein, and of assessing its purity, before you
start purification

3. Protein Purification
I. Source
II. Method of lysing the cells
III. Protecting the protein from degradation
IV. Means to keep track of your protein – activity assay,
protein purification table
V. Early Stages – gentle fractionation steps to handle
largish volumes.
VI. Middle Stages – chromatographic separation(s)
VII. Final (Polishing) Stages
Reading: N & B Chapters 6 & 7

4. I. Source
What source will you use to obtain the protein of interest?
A) Natural – some plant or animal tissue
B) Recombinant – a gene for the protein of interest that
has been cloned into a vector and transformed into
expression cells

5. Some other considerations
Structural Integrity
“native” versus “non-native” conformation
denaturation/renaturation
protein folding/re-folding
subunit dissociation/re-association

6. Choice of Buffer
The pKa of the buffer should be within 0.5 unit of the
desired pH
Potential interactions with a column matrix of any
resins you might use
Avoid UV-absorbing buffers if you plan to use a UV
detector
The ionic strength and salt composition must be
chosen according to the stability of the protein and the
detergent

7. Typical Solution Components
Buffer
Salt
Detergents (sometimes)
Glycerol, sugars
Metal chelators
Protease inhibitors (usually)
Reducing agents (often)
Ligands (sometimes)

8. Centrifuge
Be very careful using the centrifuge. Samples must
be balanced. Clean up any spills, especially those
that have ammonium sulfate, which is very corrosive.

9. On December 16, 1998, milk samples were running in a Beckman L2-65B
ultracentrifuge using a large aluminum rotor (a rotor is a large metal object
that holds the individual sample tubes and is connected to the spin drive of
the centrifuge). The rotor had been used for this procedure many times
before. Approximately one hour into the operation, the rotor failed due to
excessive mechanical stress caused by the "G" forces of the high rotation
speed. The subsequent explosion completely destroyed the centrifuge
(Images 1 & 2). The safety shielding in the unit did not contain all the metal
fragments. The half-inch thick sliding steel door on top of the unit buckled
allowing fragments, including the steel rotor top, to escape (Image 3).
Fragments ruined a nearby refrigerator and an ultra-cold freezer in addition
to making holes in the walls and ceiling. The unit itself was propelled
sideways and damaged cabinets and shelving that contained over a
hundred containers of chemicals (Image 4). Fortunately, sliding cabinet
doors prevented the containers from falling to the floor and breaking. A
shock wave from the accident shattered all four windows in the room. The
shock wave also destroyed the control system for an incubator and shook
an interior wall causing shelving on the wall to collapse (Image 5).
Fortunately the room was not occupied at the time and there were no
personal injuries.

10. We will have more to say about centrifugation, especially
analytical ultracentrifugation, during a later lecture

11. II. Method of lysing the cells
How will you “break open” the cells of the tissue or the
bacterial cells? Not a trivial matter.
A) Mechanical disruption – homogenizing the tissue with
a Waring blendor (note the spelling).
B) French Press
C) Sonication
D) Freeze Thaw Cycles
E) Enzymatic – e.g., Lysozyme or Chemical Methods

12. Frederick Waring and his “blendor”
http://extranet.libraries.psu.edu/psul/waring/blendor.html

13. A French Press, but not
the one we are interested in
http://upload.wikimedia.org/wikipedia/commons/9/91/French_press.jpg

14. This is the French Press we are interested in
Analytical Biochemistry 321 (2003) 276–277

15. Some examples of sonicators
http://en.wikipedia.org/wiki/Sonication
http://www.fishersci.com/wps/portal/PRODUCTDETAIL?aid=2819374

16. III. Protecting the protein from degradation
OK, so you have figure out a way or method to break open
the cells, now how are you going to protect your protein
from degradation?
A) Add PMSF (phenylmethylsulfonylfluoride) to inhibit
proteases
B) Add other protease inhibitors
C) High or low pH to inhibit proteases, working quickly
D) Use “Expression” cells

17. IV. Means to keep track of your protein
– activity assay, protein purification table
How are you going to know where your protein is? Is it in
the pellet or the supernatant following a centrifugation step?
A) Activity assay – a beacon in the “soup”. Activity versus
Specific Activity.
B) At each step you save a sample and later construct a
protein purification table.
C) Use of PAGE – subject for later, but not ideal as it is too
time consuming.

18. A410
N&B Fig. 7-1
Activity Assay

19. "Specific Activity"
Definition:
Units of enzyme activity per mg protein
1 Unit = amount of enzyme that will convert one
μmole of substrate to product in one minute at a
given pH (optimum value) and temperature (usually
25°C or 37°C).
Specific activity is used as an estimate of enzyme
purity.

20. Example
You have total of 5 mL of 2 mg/mL protein. From a
3 mL assay containing 1.5 mL of 50 mM substrate and
100 mL of your protein you obtain ΔA/Δt = 0.1 min-1.
Calculate the specific activity of the enzyme in the sample.
Assume that the substrate has the following extinction
coefficient: e410 nm = 18.8 mM-1 cm-1. Report your answer
to one significant figure.

21. Activity vs. Specific Activity

22. Purification Table (example)
(see N&B p. 172 for more details)

23. V. Early Stages – gentle fractionation steps to handle
largish volumes.
How can I gently separate my protein from a number of
other contaminants using relatively gentle methods?
A) Salting in/Salting out – the use of ammonium sulfate
to gently fractionate your protein
B) How to “desalt” your protein following ammonium
sulfate precipitation?
- Use of gel-filtration in the course mode
- Use of dialysis

24. Perturbations of the solvent-protein
interactions
Major forces within a polypeptide chain and
between chains that drive a protein to a stable
conformation
-Ion-ion
-Ion-dipole (hydrogen bonds)
-Hydrophobic
Perturbations of the solvent-protein interactions
can cause transitions by disrupting the “old”
interactions and promoting formation of new
ones.

25. Perturbations that cause transitions
A rise in temperature (can weaken the strength
of dipolar interactions and favor formation of
hydrophobic interactions)
pH-dependent protonation and deprotonation
Large amount of a salt or organic solvents

26. Precipitation of proteins by salts
Precipitation occurs by:
-neutralization of surface charges by the salt
-reducing the chemical activity of the protein
-diminishing the effective concentration of
water
This is called “salting out” of proteins

27. Principle behind salting out of proteins
1. The concentration of any salt necessary to cause
precipitation of a particular protein is related to the number
and distribution of charges and of hydrophobic residues
exposed and rendered dominant as the charges are
neutralized.
2. This property is exploited to separate some proteins from
others by precipitation at high salt concentrations.
3. As salt concentration increases, protein solubility
decreases.
4. Net effect is dehydration of proteins which promotes self-association
and aggregation

28. Ammonium sulfate precipitation
©Wilbur H. Campbell, 1996

29. Ammonium Sulfate Fractionation
Why use ammonium sulfate (NH4)2SO4 as opposed
to other salts?
• well tolerated by most proteins
• highly soluble (up to ~ 4 M)
• does alter pH significantly
• relatively inexpensive

31. Equilibrium Dialysis

32. VI. Middle Stages – chromatographic separation(s)
What chemical properties of your protein can you take
advantage of? What is the pI of your protein?
A) Ion-Exchange Chromatography
-anion exchange
-cation exchange
B) Affinity Chromatography
-resin contains ligand or substrate covalently attached
-use of an affinity tag (e.g., “His-tag”)

33. Affinity Chromatography
The basic idea is to use a resin that contains a
molecular fragment that interacts specifically with
the species of interest (or a relatively small class of
compounds that have similar chemical properties).
The species of interest binds to the affinity resin
while the other compounds present in the mixture
pass through the column. The species of interest
can then be eluted.

34. Affinity Chromatography Illustrated

35. Affinity Chromatography:
another view

36. Affinity Chromatography Continued: Purification of
His-tagged proteins
-from “The QIAexpressionistTM”, by Qiagen, 2003

37. Two different types of nickel
affinity resins:
NTA – nitrilotriacetic acid
IDA – iminodiacetic acid
-from “The QIAexpressionistTM”, by Qiagen, 2003

38. Binding of the His-tagged protein to the Ni-NTA
resin.
-from “The QIAexpressionistTM”, by Qiagen, 2003

39. VII. Final (Polishing) Stages
Are there still some nagging contaminants present?
A) Rerunning an ion-exchange column with a slightly
different matrix but same functional group
B) Gel-filtration chromatography – high resolution mode
C) Ultra-filtration/centrifugal concentrators

40. Ultrafiltration
Use centrifugation to
achieve solvent flux
through the membrane
Applications:
-Desalting and buffer
exchange
-Concentrating and
purifying proteins,
antibodies and nucleic
acids (alternative to
EtOH precipitation)

41. Centrifugal Concentrators
Centrifugation forces buffer through the membrane,
retaining molecules larger than the cut-off within the
sample tube. Repeated dilution and concentration can
achieve rapid buffer exchange.

42. Fundamentals of Ligand Binding:
Ligands collide with their targets, at a rate of kon.
Usually this rate is diffusion limited and occurs about
a rate of 108 – 109 M-1 s-1.
The ligand leaves its binding site at a rate that
depends on the strength of interaction between the
ligand and the binding site. Off-rates (koff) range from
106 s-1 (weak binding) to 10-2 s-1 (strong binding).
The equilibrium constant for binding is given by:
or

43. Define the fractional saturation (Y) as follows:
But, since

44. Y

45. The binding constant of a protein for a small ligand
may be determined by equilibrium dialysis