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Protein Purification (from a lecture by Dr. Richard Burgess, University of Wisconsin, Madison, at the CSH protein purifi PowerPoint Presentation
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Protein Purification (from a lecture by Dr. Richard Burgess, University of Wisconsin, Madison, at the CSH protein purification course). Object: to separate a particular protein from all other proteins and cell components. There are many proteins (over 4300 genes in E. coli )

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slide1

Protein Purification

(from a lecture by Dr. Richard Burgess, University of Wisconsin, Madison, at the CSH protein purification course).

Object: to separate a particular protein from all other proteins and cell components

There are many proteins (over 4300 genes in E. coli)

A given protein can be 0.001-20% of total protein

Other components:

nucleic acids, carbohydrates, lipids, small molecules

Enzymes are found in different states and locations:

soluble, insoluble, membrane bound, DNA bound,

in organelles, cytoplasmic, periplasmic, nuclear

slide2

Study Question

  • You are given a shoe box full of an assortment of small objects including:
          • Ping Pong balls
          • Sugar cubes
          • Paper clips
          • 1/2” brass screws
          • Iron filings
  • 1. List the properties of each of these components that might help you fractionate them.
  • 2. Devise the most efficient method you can for getting pure paper clips.
slide3

20 Naturally-occurring Amino Acids

Acidic:

D, E, (C, Y)

Basic:

K, R, H

Hydrophobic:

I, L, V, W, F

Polar:

S, T, N, Q

Other:

G, A, M, P

slide4

Overview of Protein Purification

Types of Separations

Protein Properties

Protein Inactivation/Stabilization

slide5

Protein Analysis and Purification

Analytical Separations

Gel-electrophoresis

IEF

2D-gels

Preparative Separations

Various chromatographic methods

slide7

Main Types of Molecular InteractionsHydrogen BondsN H - - - - NN-H + N low temperature high temperatureN H - - - - O C strength is very dependent on geometry donor acceptor and distance (2.6-3.1 A)Hydrophobic Interactions (waxy residues: Ileu, Leu, Val, Phe, Trp) high salt high temperature low salt Ionic Interactions (charged residues:Asp- Glu- S- Lys+ Arg+ His+)low ionic strength high ionic strength

H

H

H

H

H2O

H

H

H

H

Cl-

Na+

...

+

-

+

-

slide8

Variables that Affect Molecular Forces

Temperature

Ionic strength

Ion type

Polarity of solvent (dielectric constant)

pH

protein properties handles for fractionation

Protein Properties - Handles for Fractionation

Size(110 Da/amino acid residue)

smallest most proteins largest

Amino acids: 30 100 1,000 15,000

MW (kDa): 3.3 11 110 1,600

Multi-subunit complexes can contain 5-30 subunits

Shape

globular (sphere) asymmetric (cigar)

Effects frictional properties, effective radius, movement through pores

Centrifuge

Gel filtration

Elutes earlier

Appears larger

Sediments slower

Appears smaller

protein properties handles for fractionation10

+

-

+

+

+

+

-

+

uniform

clustered

-

+

-

-

-

-

+

-

Protein Properties - Handles for Fractionation

Net charge

Ionizable grouppKa pH2 pH7 pH12

C-terminal (COOH) 4.0 oooooooo----------------------------------------

Aspartate (COOH) 4.5 oooooooooo-------------------------------------

Glutamate (COOH) 4.6 ooooooooooo------------------------------------

Histidine (imidazole) 6.2 +++++++++++++oooooooooooooooooooo

N-terminal (amino) 7.3 +++++++++++++++oooooooooooooooooo

Cysteine (SH) 9.3 ooooooooooooooooooooooo-----------------

Tyrosine (phenol) 10.1 oooooooooooooooooooooooooo-------------

Lysine (amino) 10.4 ++++++++++++++++++++++++oooooooo

Arginine (guanido) 12.0 ++++++++++++++++++++++++++++++o

Isoelectric point

pI = pH where protein has zero net charge

Typical range of pI = 4-9

Charge distribution

versus

protein properties handles for fractionation11

H

H

H

Protein Properties-Handles for Fractionation

hydrophobic patch

Hydrophobicity

Hydrophobic residues usually are buried internally

The number and distribution on the surface vary

Can use Hydrophobic Interaction Chromatography

Solubility

Varies from barely soluble (<mg/ml) to very soluble (>300 mg/ml)

Varies with pH, ionic strength/type, polarity of solvent, temperature

Least soluble at isoelectric point where there is least charge repulsion

Ligand and metal binding

Affinity for cofactors, substrates, effector molecules, metals, DNA

When ligand is immobilized on a bead, you have an affinity bead

separation processes that can be used to fractionate proteins
Separation Processes that can be Used to Fractionate Proteins

Separation Process Basis of Separation

Precipitation ammonium sulfate solubility

polyethyleneimine (PEI) charge, size

isoelectric solubility, pI

Chromatography gel filtration (SEC) size, shape

ion exchange (IEX) charge, charge distribution

hydrophobic interaction(HIC) hydrophobicity

DNA affinity DNA binding site

immunoaffinity (IAC) specific epitope

chromatofocusing pI

Electrophoresis gel electrophoresis (PAGE) charge, size, shape

isoelectric focusing (IEF) pI

Centrifugationsucrose gradient size shape, density

Ultrafiltrationultrafiltration (UF) size, shape

slide15

Protein Sources for Purification

Traditional natural sourcesBacteria, animal and plant tissue

Cloning recombinant proteins into overexpression vector/host systems for intracellular production (E. coli the most used)

In vitro protein synthesis Transcription/translation systems

slide18

What You Can Learn from Amino Acid Sequence1. Molecular weight of the polypeptide chain 2. Charge versus pH; Isoelectric point 3. Extinction coefficient 4. Hydrophobicity & membrane spanning regions 5. Potential modification sites 6. Conserved motifs that suggest cofactor affinityWhat You Can’t Learn from Amino Acid Sequence 1. Function 2. 3-Dimensional structure; Shape 3. Multi-subunit features 4. Ammonium sulfate precipitation properties 5. Surface features (hydrophobic patches, charge distribution, antigenic sites)Conclusion: Protein Purification is still very empirical!

slide19

Engineering Proteins for Ease of Purification and Detection

Once you have a gene cloned and can over-express the protein, you can alter protein to improve the ease of purification or detection

You can fuse a tag to the N-or C- terminus of your protein

You can decide to remove the tag or not

Basic strategies

Add signal sequence that causes secretion into culture medium

Add protein that helps the protein refold and stay soluble

Add sequence that aids in precipitation

Add an affinity handle (by far the most used is the His-tag)

Add sequence that aids in detection

slide20

CSH Protein Course -Sigma32 Purification

MW A B C D E F G A/3 B/3 D/3

225

50

35

10 kDa

bb’

s32