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The Purification and Analysis of Proteins

The Purification and Analysis of Proteins The Gray Tree Frog

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

buffer that flows through the column carrying proteins simple “home - made” column sophisticated

simple “home-made” column

that flows through the column carrying proteins simple “home - made” column sophisticated instrumentation and columns
that flows through the column carrying proteins simple “home - made” column sophisticated instrumentation and columns

sophisticated instrumentation and columns

break open cells centrifuge at high speed mainly soluble proteins insoluble cell components Ion Exchange

break open cells

break open cells centrifuge at high speed mainly soluble proteins insoluble cell components Ion Exchange chromatography

centrifuge at high

speed

break open cells centrifuge at high speed mainly soluble proteins insoluble cell components Ion Exchange chromatography

mainly soluble proteins

insoluble cell

components

high speed mainly soluble proteins insoluble cell components Ion Exchange chromatography Affinity chromatography Size

Ion Exchange chromatography

Affinity chromatography

Size Exclusion chromatography

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

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

2.18 8.95 10.79 10.79 8.95 2.18 Nelson p85
2.18
8.95
10.79
10.79
8.95
2.18
Nelson p85

pI

=

(pK 2 + pK 3 )

2

=

(10.79 + 8.95)

2

=

9.87

Lysine:

pKa carboxyl = 2.18 pKa amino = 8.95 pKa sidechain = 10.79

Notice that the calculation of pI always involves the pKa’s that bracket the neutral charge

Notice that at pH below its pI, lysine is predominantly positively charged

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

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

Arginine

17

3

Lysine

8

4

Aspartate

6

6

Glutamate

4

6

Net charge

+15

-5

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

A chromatography column Ion Exchange Medium Cation Exchange Beads Anion Exchange Beads proteins with net

A chromatography column

Ion Exchange Medium

Cation Exchange Beads

Anion Exchange Beads

Exchange Medium Cation Exchange Beads Anion Exchange Beads proteins with net (+) charge will bind to

proteins with net (+) charge will bind to these beads

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

has net negative charge negatively charged proteins bind to DEAE groups Positively charged medium flow of
has net negative charge negatively charged proteins bind to DEAE groups Positively charged medium flow of

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

Na + - Cl Na + - Cl - - Cl Cl Na + Na
Na +
-
Cl
Na +
-
Cl
-
-
Cl
Cl
Na +
Na +
-
Cl
Na +
-
Cl

This is why DEAE is called an ANION exchange medium

Na + - Cl Na + - Cl - - Cl Cl Na + Na +

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 DNA
Bead
DNA

Ligand

your own affinity chromatography medium. Bead DNA Ligand protein of interest binds ligand, all other proteins
your own affinity chromatography medium. Bead DNA Ligand protein of interest binds ligand, all other proteins
your own affinity chromatography medium. Bead DNA Ligand protein of interest binds ligand, all other proteins
your own affinity chromatography medium. Bead DNA Ligand protein of interest binds ligand, all other proteins
your own affinity chromatography medium. Bead DNA Ligand protein of interest binds ligand, all other proteins
your own affinity chromatography medium. Bead DNA Ligand protein of interest binds ligand, all other proteins

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

Mixture of large, medium, small sized proteins

Mixture of large, medium, small sized proteins small protein medium protein large protein flow of buffer

small

protein

medium

protein

large

protein

flow of buffer

Beads in the size exclusion medium contain pores small proteins enter the pores and take

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

circuitous route thru medium Large proteins are “excluded” from the pores, therefore travel faster thru the

Elution Profile from Size Exclusion Column

20 kDa 100 kDa 40 kDa time amount of protein
20 kDa
100 kDa
40 kDa
time
amount of protein
break open cells centrifuge at high speed mainly soluble proteins insoluble cell components Ion Exchange

break open cells

break open cells centrifuge at high speed mainly soluble proteins insoluble cell components Ion Exchange chromatography

centrifuge at high

speed

break open cells centrifuge at high speed mainly soluble proteins insoluble cell components Ion Exchange chromatography

mainly soluble proteins

insoluble cell

components

high speed mainly soluble proteins insoluble cell components Ion Exchange chromatography Affinity chromatography Size

Ion Exchange chromatography

Affinity chromatography

Size Exclusion chromatography

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

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

Electrophoresis) separates proteins on basis of mass/size Protein Gels use polyacrylamide a polymer that restricts

Protein Gels use polyacrylamide

a polymer that restricts protein migration in an electric field

- +
-
+
- + stain the gel to visualize proteins

stain the gel to visualize proteins

- + stain the gel to visualize proteins

Proteins are treated with detergent SDS (sodium dodecyl sulfate) and a reducing agent

before running on gel

dodecyl sulfate) and a reducing agent before running on gel structure of SDS the reducing agent

structure of SDS

and a reducing agent before running on gel structure of SDS the reducing agent β -mercaptoethanol

the reducing agent β-mercaptoethanol breaks disulfide bonds in proteins

Proteins are treated with detergent SDS (sodium dodecyl sulfate) and a reducing agent

before running on gel

-
-

Beta-mercaptoethanol

(reducing agent)

running on gel - Beta-mercaptoethanol (reducing agent) SDS - S S - - - - -

SDS

- S S - - - - - - - - - - - -
-
S
S
-
-
-
-
- -
-
-
-
-
-
-
-
-

proteins are fully unfolded, coated in uniform negative charge thus separate on the basis of size only

kDa The Mass of Proteins Protein mass is usually reported as kDa (kiloDaltons) The Dalton

kDa

kDa The Mass of Proteins Protein mass is usually reported as kDa (kiloDaltons) The Dalton is

The Mass of Proteins

Protein mass is usually reported as kDa (kiloDaltons)

The Dalton is an atomic mass unit

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