The Purification and Analysis of Proteins

The Gray Tree Frog

 Today we will consider just a few of the techniques associated with

purifying proteins from cells.

Purifying a protein away from all other cellular components allows us to

understand the structure and function of proteins and their roles
in living systems
The principles we have learned so far are relevant to the
techniques used in protein purification

Purification of Proteins from Cells

Cells usually contain many thousands of different proteins all involved
in the business of contributing and maintaining cell structure and catalysing
the chemical reactions that define life.
The bacterium Escherichia coli has about 4,100 protein coding genes in
its genome.
Humans have about 23,000 protein coding genes.
Not all of these proteins are expressed at any given time, and for those that
are being expressed in the cells, some are highly expressed (the protein is
very abundant in the cells) and some are expressed to low levels (and there
are not many copies of the protein per cell).
One might wish to purify a specific protein away from all other proteins and cell
materials for a variety of reasons:
-structural studies (e.g. X-ray crystallography requires pure protein)
-the analysis of enzymatic function is best accomplished with pure protein

General Scheme for purifying a given protein

1. Obtain enough cells containing enough of the protein of interest.
2. Disrupt the cells so that all proteins in the cytoplasm are liberated.
3. Use a series of separation techniques (chromatography techniques) that exploit
differential properties of your protein of interest (size, charge on
your protein).
4. Monitor the progress and success of your purification scheme using gel
electrophoresis and protein staining techniques.

Three Main Chromatography Techniques

Chromatography means to pass a mixture of proteins, which are carried in a
buffer solution, through a porous column of small beads that either separate
proteins on the basis of size or bind certain proteins on the basis of their
chemical characteristics.
Ion Exchange Chromatography
separates proteins on the basis of their charge

Affinity Chromatography
separates proteins on the basis of the ability of certain proteins to bind
certain other molecules (ligands)
Size Exclusion Chromatography
separates proteins on the basis of their size.

Column Chromatography consists of three elements:

1) a column
2) a particulate medium (beads) packed into column
3) a liquid buffer that flows through the column carrying proteins

simple home-made column

sophisticated instrumentation
and columns

break open cells

centrifuge at high
speed

mainly
soluble proteins

insoluble cell
components

Ion Exchange chromatography

Affinity chromatography

Size Exclusion chromatography

monitor successive
purification at
each step using gel
electrophoresis and
protein staining

Ion Exchange Chromatography

Separation on the basis of protein charge

Like amino acids, proteins have a specific pI and a

net charge at a certain pH
Lysine:
2.18

10.79

8.95

pKacarboxyl = 2.18
pKaamino = 8.95
pKasidechain = 10.79

10.79
8.95

Notice that the calculation of pI

always involves the pKas that
bracket the neutral charge
2.18

Notice that at pH below its

pI, lysine is predominantly
positively charged

Nelson p85

(pK2 + pK3)
pI =

(10.79 + 8.95)
=

= 9.87

 At pH 7, the amino acids glutamate and aspartate are negatively charged

and the amino acids arginine and lysine are positively charged
Proteins are composed of hundreds or thousands of amino acids and they
have a characteristic isoelectric point (pI) and carry an overall net charge
depending on the pH of the medium and the abundance
of the charged amino acids in the protein.
Proteins that have an pI > 7 will have a net positive charge at pH 7 and
are called basic proteins
Proteins that have an pI < 7 will have a net negative charge at pH 7 and
are called acidic proteins
Most proteins therefore will either be negatively charged or positively charged
at pH 7 and this fact can be used to separate (purify) them from each other

**of course a minority of proteins will have an pI = 7 and they will be neutral
at pH 7.

Ion Exchange Chromatography

Every protein has a net charge at pH 7 which depends on the number of
positively charged amino acids (Arginine and Lysine) and the number of
negatively charged amino acids (Glutamate and Aspartate).
For example, consider two proteins that amongst all of their amino acids have:
Protein 1

Protein 2

17

Lysine

Aspartate

Glutamate

Net charge

+15

-5

Arginine

These net charges can help purify a target protein away from other cellular proteins
using ion-exchange chromatography.

Ion Exchange Medium

Cation Exchange Beads Anion Exchange Beads

proteins with net (+)

charge will bind to
these beads

A chromatography column

proteins with net (-)

charge will bind to
these beads

Mixture of positively charged and negatively charged proteins

Target protein has net negative charge

negatively charged proteins

bind to DEAE groups

Positively
charged
medium

flow of buffer

Proteins bound to beads can be released by adding other negative charged ions
that will compete with protein for binding the beads
NaCl is used because the negatively charged Chloride ions will act as competitor

Cl

Na+

Cl

Na+

Na+

Cl

Cl

Na+
Cl

Na+
Cl

This is why
DEAE is called
an ANION
exchange medium

Affinity Chromatography
affinity chromatography relies on fact that proteins, as part of their function, bind
other molecules called Ligands.
For example, some proteins that help regulate gene expression bind double-stranded
DNA at a specific sequence of nucleotides.
If you are trying to purify such a protein, it would be easy to make your own
affinity chromatography medium.

Bead

Ligand
DNA

protein of interest binds

ligand, all other proteins
pass through.

Size Exclusion (Gel Filtration)

separates proteins on the basis of protein size (measured in kiloDaltons (kDa))
like other chromatography media, uses very small beads
a mixture of different sized proteins is added to top of column
proteins migrate thru medium at different rates
large proteins travel faster thru column than small proteins
proteins exit the column at different times and are collected in tubes

Mixture of large, medium, small sized proteins

small
protein

medium
protein

large
protein

flow of buffer

Beads in the size exclusion

medium contain pores
small proteins enter the pores
and take circuitous route thru
medium
Large proteins are excluded
from the pores, therefore
travel faster thru the medium

amount of protein

Elution Profile from Size Exclusion Column

20 kDa

100 kDa

40 kDa

time

break open cells

centrifuge at high
speed

mainly
soluble proteins

insoluble cell
components

Ion Exchange chromatography

Affinity chromatography

Size Exclusion chromatography

monitor successive
purification at
each step using gel
electrophoresis and
protein staining

Electrophoresis of Proteins in Gels

SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) separates
proteins on basis of mass/size

Protein Gels use polyacrylamide

a polymer that restricts protein
migration in an electric field

+
stain the gel to
visualize proteins

Proteins are treated with detergent SDS (sodium dodecyl sulfate) and a reducing agent
before running on gel

structure of SDS

the reducing agent -mercaptoethanol breaks disulfide bonds in proteins

Proteins are treated with detergent SDS (sodium dodecyl sulfate) and a reducing agent
before running on gel

SDS

Beta-mercaptoethanol
(reducing agent)

S S

proteins are fully unfolded, coated in uniform negative charge

thus separate on the basis of size only

The Mass of Proteins

Protein mass is usually reported as kDa (kiloDaltons)
The Dalton is an atomic mass unit
kDa

One Dalton is equal to 1/12 the mass of the Carbon12 atom.

So,

a carbon atom has a mass of 12 Daltons

a hydrogen atom has a mass of 1 Dalton

The average mass of an amino acid is 110 Daltons

therefore, a 318 amino acid protein would have a mass of
318 x 110 = 34,980 Da = 35 kDa

Reminder
Midterm 1 Wed Oct 14

bring pencil, eraser, calculator

multiple choice, scantron
-exams will be placed under each chair
-do not enter room until directed by invigilators