Lecture 3: Purification of Proteins

Deborah Clark
Quinnipiac University
Bi515, 2/2/10

Peptide synthesis, read for overview but don’t memorize structures

Problems in text Chapter 3 4th edition: 14,15,18
(5th edition 10, 11 and 15 (see problem solving disk!), 17-19, 22, 23 )
 Purification of Proteins

 Isolate from cell in native conditions

 Need enough starting material—usually isolate organelle
(eukaryote)
 Multiple steps, some designed for handling large
volume/bulk, others relying on specific properties of
proteins
 Need an assay to follow where protein of interest is.
Monitor specific activity: protein activity/mg protein
 Try to minimize denaturation: inhibitors of proteases, cold,
glycerol (CH2OHCH2OHCH2OH) may simulate more
organic environment around proteins (not all surrounded by
just water)
General Strategy for Protein Purification
Homogenization/extraction

Differential centrifugation

Salting-out/dialysis and concentration

Ion-exchange and gel-filtration chromatography (or other)

SDS-PAGE

Proteolytic degradation

Peptide purification and sequencing

Purification, cont.
 Add -mercaptoethanol or dithiothreitol to reduce disulfide
bonds
 SHCH2CH2OH or ring structure with S-S (DTT). Problem
solving disk,
question 10
shows this
quite well!

OH OH
Reduction of
Disulfide Bond
C C
H2C CH2
S S
X-S-S-Y X-SH
+ HS-Y
Reduced form Oxidized form
Reduction and Acetylation of Disulfide Bonds for
Peptide Sequencing—another application of
disulfide reducing agents
EDTA
Chelator of divalent cations
Helps break down cytoskeleton
May cause aggregation of proteins or activate their
degradation by enzymes
 First step: Breaking cells
Second step: centrifugation

Fc (centrifugal force)=m’2r = m(1-p)  2r

m’=effective mass (<mass) to account for buoyancy factor
=partial specific volume=1/density
P=density of solution
=angular velocity (set rotor speed)
r=radius of rotor

Sedimentation velocity of protein = Fc/f

(directly proportional to strength of centrifugal field (Fc))
f=frictional drag on particle
The higher the s value, the larger/more globular the protein
 Differential Centrifugation
Cells (eukaryote) 1000 x g x 5’

Chloroplasts, nuclei 4000 x g x 10’

Mitochondria, bacteria, 10,000 x g x 20’

lysosomes, peroxisomes
Ribosomes, microsomes 100,000 x g x 3 hr
Ammonium Sulfate Precipitation
(Salting In/Out) (handout)

Increase protein-protein interactions

Decrease protein-solvent interactions
Proteins tend to be least soluble at pI, so aggregate
with each other, and eventually precipitate (“salt out”).
Other proteins become less soluble (they “salt in”).
At pI, mostly uncharged, so don’t repel each other

For proteins that tend to “salt out”, at what pH do you think they are
least soluble: above, below or at their isoelectric point?
Why precipitate proteins—to concentrate them
Precipitation with Ammonium
Sulfate (handout)
Principle of ammonium sulfate precipitation
 Different Proteins Precipitate Out With Different Concentrations of
Ammonium Sulfate
How do we separate the different proteins?
Add ammonium sulfate slowly with stirring, then use differential
centrifugation to pellet those proteins that precipitated

o 15% Pellet e.g. If ammonium sulfate is added to

33%, any protein that requires 45%
NH4SO4
o 33% or 60% to precipitate will still be in
added o 45% Supernatant the supernatant. What proteins will
be in the pellet?
o 60%
 Column Chromatography

Mobile phase,
solid phase (resin)

3 proteins
separated
 Cation Exchange Chromatography

Two types of
Ion exchange:
--anion
--cation

These columns
often used first in
purification--why??
 Ion Exchange Chromatography
Cation Exchange Anion Exchange
CM-cellulose [carboxymethyl cellulose] (-) DEAE cellulose [dimethylaminoethyl cellulose] (+)

http://www.imb-jena.de/~rake/
http://irfanchemist.wordpress.com/2009/04/19/purificati Bioinformatics_WEB/proteins_purification.ht
on-of-protein/ ml
 Quiz Yourself!
__-charged proteins bind to anion exchange chromatography
resin, and need to be eluted with __ ions (Cl- or Na+), while
___-charged proteins flow through an anion exchange column.
Which amino acids would bind to an anion exchange column at
neutral pH? Aspartate? Glutamine? Phenylalanine? Lysine?

__-charged proteins bind to a cation exchange chromatography

resin, and need to be eluted with __ ions (Cl- or Na+), while
___-charged proteins flow through a cation exchange column.
Which amino acids would bind to a cation exchange column at
neutral pH? Aspartate? Glutamine? Phenylalanine? Lysine?
Ion Exchange Chromatography: anion, cation
 See animation at
http://www.wiley.com/college/fob/quiz/quiz05/5-5.html
Change the salt concentration to elute bound
proteins
ions in salt bind to the protein and neutralize it's charge, so
that the protein is released from the gel.
 Can also change the pH to elute bound proteins
(see next slide)
Changing the pH to elute bound proteins:
crude separation according to the isoelectric point (pI) - the pH at which a protein has no net
charge. (Remember - proteins become more positively charged as the pH decreases due to
the high H+ concentration.) Problem
solving
disk,
Q11

http://www.bch.bris.ac.uk/staff/pfdg/teaching/purify.htm
Separation of Amino Acids by Ion-Exchange
Chromatography
 Amino acids eluted from a column in the following order (from first to elute to
last):

(1) Asp Thr Ser Glu Pro Gly Ala

Val Met Ile Leu Tyr Phe Lys

His Arg (16)

 What type of resin was contained in the column?

 How many types of interactions does this resin have with the amino acids?

 The cation-exchange resin contains sulfonate groups bound to a hydrophobic

polystyrene resin. Elution depends on the interaction of the a.a. side chain with the
sulfonate groups (negatively charged) as well as the polystyrene resin (nonpolar).
Gel Filtration Problem
solving
disk,
Chromatography (sizing) is Q10

usually last

Why?
Note large proteins elute
first

Hydrophobic
chromatography also
available

Gel Filtration (sizing chromatography)

http://www.wiley.com/college/fob/quiz/quiz05/5-6.html
Affinity
Chromatography
is the best!
(1000x)
Two Examples of Affinity Chromatography
(from Stryer text)

http://www.imb-jena.de/~rake/Bioinformatics_WEB/proteins_purification.html
HPLC (High Performance Liquid Chromatography)

http://www.forumsci.co.il/HPLC/modes/modes.gif
 HPLC
 High pressure, buffer flow controlled by pumps
 Small volumes of sample needed
 Very fast
 Analytical or for purification of small amounts
 What types of molecules? Small! (peptides, small sugars,
nucleotides)
 Separation according to affinity for stationary vs. mobile phase
(reverse-phase, normal phase HPLC)
 Application: drug testing in urine of athletes, horses, dogs,
pregnancy testing
HPLC Notes
Columns
Reversed-phase (most common)
 Water/organic mobile phase
 Nonpolar organic stationary phase, usually on silica surface
 Chemistry: hydroxylated silica surface-OH + X-Sl-(CH3) 2-R,
where R = C-18 (most common), C-8, CN or NH2
Normal-phase
 Nonpolar mobile phase
 Polar stationary phase
Ion-pair (like ion exchange)
Size-exclusion
FPLC (Fast performance liquid
chromatography)
FPLC
Buffer flow controlled by pumps
Liquid chromatography columns of all types
Not as fast as HPLC
Used for purification of proteins
Computer control allows for ease of purification,
better control
Check for purity by Sodium dodecylsulfate
polyacrylamide gel electrophoresis (SDS-PAGE )

SDS-PAGE Animation
Migration proportional to strength of electric
field, net charge on protein, frictional coefficient

E:\Chapter Resources\chapter 03\animations\03_SDS_gel_electro.html

Example of Protein Purification
 Problem
solving
disk,

SDS-PAGE Q10

Try the Simulation at

http://people.rit.edu/pac8612/electro/Electro_Si
m.html

Effect of Acrylamide concentration

Lower % acrylamide
Higher % acrylamide
Plotting migration (Rf) of Standards
and determining MW of unknown
Isoelectric focusing to determine pI or to
purify using pI

pH gradient
formed in gel
Isoelectric Focusing (Analytical)
 Separation in a non-denaturing acrylamide gel (no SDS)
according to the isoelectric point (pI) of the protein

 Proteins in their native state. Electrophoresis is in a

polyacrylamide gel with a pH gradient. Proteins migrate in gel
to a point where the pH matches their pI; then they stop as they
have zero net charge (and are unaffected by the electric field).

 An acidic protein (negative charge at pH 7) will travel towards

the end with lower pH and positive charge, until the pH equals
it's own pI. Explain this in terms of changes in functional
groups.

Low pH High pH,

+ Electrode - Electrode
2-Dimensional Gel Electrophoresis (3-22)
 2D-gel electrophoresis
Separation of proteins by isoelectric focusing (in
one lane, or in tube gel)
Placement of tube gel onto SDS-PAGE gel
Separation of proteins by MW in SDS-PAGE
This method is powerful enough to positively
resolve over 1000 proteins from a total protein
sample of E.Coli, and is sometimes used in
forensics to analyse samples and determine their
source.

http://www.bch.bris.ac.uk/staff/pfdg/teaching/purify.htm
Group Problem:
Phosphorylation of protein C depends on the temperature at which the cells
are grown.
 
Purified protein C from cells grown at 37 ºC and purified protein C from cells
grown at 25 ºC are subjected to two-dimensional PAGE:
1) isoelectric focusing followed by 2) SDS-polyacrylamide gel electrophoresis.

1
(high pH) IEF (low pH)

2
SDS-PAGE

protein C from protein C from

cells grown at 25 °C cells grown at 37 °C
(high pH) IEF (low pH)

SDS-PAGE

protein C from protein C from

cells grown at 25 °C cells grown at 37 °C

 Is protein C phosphorylated in cells grown at 37 ºC or at 25

ºC? Explain the reasoning behind your answer.

 Why are the migrations of protein C from cells grown at 37

ºC and 25 ºC identical on SDS-PAGE?

 What is another method that could be used to determine at

which temperature protein C is phosphorylated?
 Problem 15 (Ed 5; 14 Ed 4). Purification of an enzyme
Problem
solving Procedure Total protein Activity (units) Specific
disk (mg) Activity
(units/mg)
1. Crude extract 20,000 4,000,000 200
2. Precipitation (salt) 5,000 3,000,000 600
3. Precipitation (pH) 4,000 1,000,000 250
4. Ion-exchange chrom. 200 800,000 4000
5. Affinity chrom. 50 750,000 15000
6. Size-exclusion chrom. 45 675,000 15000

1. Calculate the specific activity of the enzyme solution after each step
of the purification. (Go over specific activity)
2. Which of the procedures was most effective (i.e. gave the greatest
relative increase in purity)?
3. Which of the procedures was least effective?
4. Is there any indication that the enzyme after step 6 is now pure?
What else could be done to estimate the purity?
Protein Structure—automated amino acid sequencing (5-25)

PTH-
derivative
 Good for about 30 residues. Large peptides must be cleaved first,
separated by HPLC, then sequenced. Repeat using different cleavage
enzyme for overlaps.

Table 3-7

Thermolysin
Cleavage points:
Nonpolar AAs (N)
Cleaving proteins and sequencing and ordering the peptide fragments (3-27)
Protein homology among species

•Each row=different species, comparing same protein

•Yellow=identical aa=invariant
•Blue=conservative substitutions (same aa family)
•Unshaded=nonconservative substitutions (diff. aa family)
•Helpful for determining function of unknown protein
•Can use nucleotide sequencing to get same result
Group Exercise
Which of the following is a conservative substitution?
Val for Ile
His for Pro
Asp for Ala
Lys for Leu
Ser for Ala

#5 Baggott and Angstadt

Electrospray Ionization Mass Spectrometry (ESI-MS)
box 5-3

Proteins dispersed into droplets using needle and high voltage

electric field. Droplets evaporate and are charged. The charged
droplets migrate in electric and/or magnetic field, depending on
their m/z (mass/charge) ratio. MALDI-MS (matrix assisted laser
desorption/ionization MS) uses laser light to ionize proteins which
then go into vacuum.
Electrospray Ionization Mass Spectrometry

Each peak from right to left is +1 more in charge. Transform

spectrum into one peak with protonated mass. Highly accurate.
Why sequence or determine mass?
Search databases for known functions
Or, search for domains in proteins, e.g. ATP binding
site
Signal sequences determine location in cell
Other sequences are attachments sites for fatty acid,
carbohydrate, or other ligand
 Determine the positions of the disulfide bonds
and the primary sequence of the polypeptide
1. Upon reduction of the disulfide bonds, two peptide subunits are obtained
with the following sequnces:

1. Chain 1: Ala-Cys-Phe-Pro-Lys-Arg-Trp-Cys-Arg-Arg-Val-Cys
2. Chain 2: Cys-Tyr-Cys-Phe-Cys

2. The native polypeptide (intact disulfide bonds) is digested with

thermolysin and the following peptides are obtained (results are after
acid hydrolysis of the peptides):

Thermolysin
1. (Ala, Cys2, Val)
Cleavage points:
2. (Arg, Lys, Phe, Pro)
Nonpolar AAs (N)
3. (Arg2, Cys2, Trp, Tyr)
(not proline)
4. (Cys2, Phe)
Wood, Wilson, Benbow and Hood (1974) Biochemistry:
A Problems Approach (#3-7)
Group Exercise
Determine the amino acid sequence of this peptide

 Complete hydrolysis by 6 M HCl at 110 C followed by a.a. analysis indicated

the presence of Ala, Ser, Lys, Phe, Trp and Met in a 2:1:1:1:1:1 ratio.

 Treatment of the peptide with 1-fluoro-2,4-dinitrobenzene followed by

complete hydrolysis and chromatography indicated the presence of 2,4-DNP-
Alanine.

 Complete digestion of the peptide with the treatments below, followed by

acid hydrolysis and chromatography yielded peptides as listed.

Chymotrypsin (Phe,Trp,Tyr (C)) CNBr (Met (C))

(Phe, Ala) (Ala, Trp)
(Ser, Lys, Trp, Met) (Phe, Lys, Ala, Ser, Met)
(Ala)
Trypsin (Lys,Arg ( C))
(Phe, Lys, Ala)
(Ser, Trp, Ala, Met)