8/18/2019 Lecture 10- Protein Analysis

http://slidepdf.com/reader/full/lecture-10-protein-analysis 1/27

The Purification and Analysis of Proteins

The Gray Tree Frog

8/18/2019 Lecture 10- Protein Analysis

http://slidepdf.com/reader/full/lecture-10-protein-analysis 2/27

• 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 tounderstand 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

8/18/2019 Lecture 10- Protein Analysis

http://slidepdf.com/reader/full/lecture-10-protein-analysis 3/27

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

8/18/2019 Lecture 10- Protein Analysis

http://slidepdf.com/reader/full/lecture-10-protein-analysis 4/27

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 onyour protein).

4. Monitor the progress and success of your purification scheme using gel

electrophoresis and protein staining techniques.

8/18/2019 Lecture 10- Protein Analysis

http://slidepdf.com/reader/full/lecture-10-protein-analysis 5/27

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 separateproteins 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.

8/18/2019 Lecture 10- Protein Analysis

http://slidepdf.com/reader/full/lecture-10-protein-analysis 6/27

simple “home-made” column 

sophisticated instrumentation

and columns

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

8/18/2019 Lecture 10- Protein Analysis

http://slidepdf.com/reader/full/lecture-10-protein-analysis 7/27

break open cells

centrifuge at high

speed insoluble cell

components

mainly

soluble proteins

Ion Exchange chromatography

Affinity chromatography

Size Exclusion chromatography

monitor successive

purification at

each step using gelelectrophoresis and

protein staining

8/18/2019 Lecture 10- Protein Analysis

http://slidepdf.com/reader/full/lecture-10-protein-analysis 8/27

Ion Exchange Chromatography

Separation on the basis of protein charge

8/18/2019 Lecture 10- Protein Analysis

http://slidepdf.com/reader/full/lecture-10-protein-analysis 9/27

2.18

8.95

10.79

10.798.952.18

Nelson p85

Lysine:

pKacarboxyl = 2.18

pKaamino = 8.95

pKasidechain = 10.79

pI =2

(pK2 + pK3) 

=2

(10.79 + 8.95) 

= 9.87

Like amino acids, proteins have a specific pI and a

net charge at a certain pH

Notice that at pH below its

pI, lysine is predominantly

positively charged

Notice that the calculation of pI

always involves the pKa’s that

bracket the neutral charge

8/18/2019 Lecture 10- Protein Analysis

http://slidepdf.com/reader/full/lecture-10-protein-analysis 10/27

• 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 andare 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.

8/18/2019 Lecture 10- Protein Analysis

http://slidepdf.com/reader/full/lecture-10-protein-analysis 11/27

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:

These net charges can help purify a target protein away from other cellular proteins

using ion-exchange chromatography.

Protein 1 Protein 2

Arginine 17 3Lysine 8 4

Aspartate 6 6

Glutamate 4 6

Net charge +15 -5

8/18/2019 Lecture 10- Protein Analysis

http://slidepdf.com/reader/full/lecture-10-protein-analysis 12/27

A chromatography column

Ion Exchange Medium

Cation Exchange Beads Anion Exchange Beads

proteins with net (+)

charge will bind to

these beads

proteins with net (-)

charge will bind to

these beads

8/18/2019 Lecture 10- Protein Analysis

http://slidepdf.com/reader/full/lecture-10-protein-analysis 13/27

flow of buffer

Mixture of positively charged and negatively charged proteins

Target protein has net negative charge

Positively

chargedmedium

+ -

negatively charged proteins

bind to DEAE groups

+

8/18/2019 Lecture 10- Protein Analysis

http://slidepdf.com/reader/full/lecture-10-protein-analysis 14/27

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-Cl-

Cl-

Na+

Cl-Na+

Na+

Na+

Na+

Cl-

Cl-

This is why

DEAE is called

an ANION

exchange medium

8/18/2019 Lecture 10- Protein Analysis

http://slidepdf.com/reader/full/lecture-10-protein-analysis 15/27

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.

8/18/2019 Lecture 10- Protein Analysis

http://slidepdf.com/reader/full/lecture-10-protein-analysis 16/27

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

8/18/2019 Lecture 10- Protein Analysis

http://slidepdf.com/reader/full/lecture-10-protein-analysis 17/27

8/18/2019 Lecture 10- Protein Analysis

http://slidepdf.com/reader/full/lecture-10-protein-analysis 18/27

small

protein

medium

protein

large

protein

flow of buffer

Mixture of large, medium, small sized proteins

8/18/2019 Lecture 10- Protein Analysis

http://slidepdf.com/reader/full/lecture-10-protein-analysis 19/27

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

8/18/2019 Lecture 10- Protein Analysis

http://slidepdf.com/reader/full/lecture-10-protein-analysis 20/27

time

100 kDa 40 kDa

20 kDa

   a   m   o   u   n   t   o    f   p   r   o   t   e   i   n

Elution Profile from Size Exclusion Column

8/18/2019 Lecture 10- Protein Analysis

http://slidepdf.com/reader/full/lecture-10-protein-analysis 21/27

8/18/2019 Lecture 10- Protein Analysis

http://slidepdf.com/reader/full/lecture-10-protein-analysis 22/27

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

8/18/2019 Lecture 10- Protein Analysis

http://slidepdf.com/reader/full/lecture-10-protein-analysis 23/27

stain the gel to

visualize proteins

+

-

8/18/2019 Lecture 10- Protein Analysis

http://slidepdf.com/reader/full/lecture-10-protein-analysis 24/27

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

before running on gel

structure of SDS

the reducing agentβ

-mercaptoethanol breaks disulfide bonds in proteins

P t i t t d ith d t t SDS ( di d d l lf t ) d d i t

8/18/2019 Lecture 10- Protein Analysis

http://slidepdf.com/reader/full/lecture-10-protein-analysis 25/27

S S

-

-

-

--

-

--

- - -

--

-

-

-SDS

Beta-mercaptoethanol

(reducing agent)

proteins are fully unfolded, coated in uniform negative charge

thus separate on the basis of size only

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

before running on gel

8/18/2019 Lecture 10- Protein Analysis

http://slidepdf.com/reader/full/lecture-10-protein-analysis 26/27

kDa

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

8/18/2019 Lecture 10- Protein Analysis

http://slidepdf.com/reader/full/lecture-10-protein-analysis 27/27

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