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Protein Purification. Compartmentalization provides an opportunity for a purification step. e. Protein profile for compartments of gram-negative prokaryotes. Cell Disruption. Chemical: alkali, organic solvents, detergents

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slide2
Compartmentalization provides an opportunity

for a purification step

e

Protein profile for compartments of gram-negative prokaryotes

cell disruption
Cell Disruption
  • Chemical: alkali, organic solvents, detergents
  • Enzymatic: lysozyme, glucanases, chitinase
  • Physical: osmotic shock, freeze/thaw
  • Mechanical: sonication, homogenization, wet milling, French press
chemical disruption
Chemical Disruption

Detergents such as Trition X-100 or NP40 can permeabilize cells by solubilizing membranes.

Detergents can be expensive, denature proteins, and must be removed after disruption

french press
French Press

Cells are placed in a stainless steel container. A tight fitting piston is inserted and high pressures are applied to force cells through a small hole.

homogenization
Homogenization

Cells are placed in a closed vessel (usually glass). A tight fitting plunger is inserted and rotated with a downward force. Cells are disrupted as they pass between the plunger and vessel wall. Also, shaking with glass beads works, BUT:

Friction = Heat

sonication
Sonication

A sonicator can be immersed directly into a cell suspension. The sonicator is vibrated and high frequency sound waves disrupt cells.

slide9
Inclusion bodies provide a rapid purification step

Inclusion bodies provide

storage space for protein,

carbohydrate and lipid

material in prokaryotes

However, proteins exist

as aggregates in inclusion

bodies thus special

precautions must be taken

during purification

slide10
10%

Glycerol

40%

Glycerol

Even proteins can be separated by their

sedimentation properties

Function of both

size and shape

slide11
Proteins have unique properties resulting

from their amino acid composition

  • Localization
  • Charge
  • Hydrophobicity
  • Size
  • Affinity for ligands

Arbitrary protein

slide12
The charge on a protein is dependent upon pH
  • The content of amino acids with ionizable

side chains determines the overall charge

of a protein

  • Thus, a protein containing a majority of basic
  • residues (ie. R and K) will be positively charged
  • and will bind to a cation-exchange support

Ion exchange column

Supports (examples)

slide14
Cation exchange chromatography
  • Protein samples are applied to

this column at low ionic strength,

and positively charged proteins

bind to the column support

  • Proteins are eluted using a gradient

of increasing ionic strength, where

counterions displace bound protein,

changing pH will also elute protein

  • Choice of functional groups on

distinct column supports allow a

range of affinities

Na+Cl-

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Protein

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slide15
Conversely, at a pH two orders of magnitude

above their pKa, acidic amino acids will be

negatively charged, thus proteins with a majority of

acidic amino acids (D and E) will be negatively

charged at physiological pH

  • Negatively charged proteins can be separated using
  • anion exchange chromatography
slide16
Anion exchange chromatography
  • Protein samples are applied to

this column at low ionic strength,

and negatively charged proteins

bind to the column support

  • Proteins are eluted using a gradient

of increasing ionic strength, where

counterions displace bound protein,

changing pH will also elute protein

  • Choice of functional groups on

distinct column supports allow a

range of affinities

  • Bead size affects resolution in both

anion and cation exchange

Na+Cl-

Protein

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

slide17
Hydrophobic Interaction Chromatography
  • Although most hydrophobic amino acids are buried in
  • the interior of proteins, many proteins have hydrophobic
  • surfaces or patches which can be used for separation
  • A protein’s hydrophobic character is typically enhanced
  • by addition of high salt concentrations
  • Proteins are eluted from HIC columns via a gradient of
  • high salt to low salt concentrations
isoelectric focusing
Isoelectric Focusing

For any protein, there is a characteristic pH at which the protein has no net charge (isoelectric point).

At the isoelectric pH, the protein will not migrate in an electric field.

protein precipitation
Protein Precipitation
  • Precipitation is caused by changes that disrupt the solvating properties of water
  • Changes in pH, ionic strength, temperature, and the addition of solvents can cause precipitation (loss of solubility)
  • Most proteins have a unique set of conditions that result in precipitation
precipitation with salt
Precipitation with Salt
  • In practice, most procedures use the salt ammonium sulfate (NH4)2SO4 to precipitate proteins
  • The amount of salt required is directly related to the number and distribution of charged and nonionic polar amino acids exposed on the surface of the protein
slide22
Salt effects on protein solubility

At low ionic strengths, the charges on the surface of a protein attract counter ions, decreasing electrostatic free energy and increasing solubility. Addition of low concentrations of salt, then, increase solubility of proteins ("salting in"). At high salt concentrations, however, protein solubility decreases ("salting out"). This is due to electrostatic repulsion between the surface ions and the hydrophobic interior of the protein and to the avid interaction of salts with water. This disrupts the ordered water in the hydration layer. Salts vary in their ability to salt out proteins and generally follow the Hofmeister series:

Cations: NH4+ > K+ > Na+ > Mg++ > Ca++ > guanidium

Anions: SO4-- > HPO4-- > acetate > citrate > tartrate > Cl- > NO3-

slide24
Proteins can be separated on the basis of size
  • Gradient centrifugation
  • Gel filtration
slide25
Gel Filtration provides a molecular sieve

Figures from Scopes, Protein Purification

on Reserve

gel filtration chromatography
Gel Filtration Chromatography

Proteins that enter porous beads will migrate slower than proteins that are excluded from the pores.

Separation is a function of relative size and shape

size exclusion can be used to determine oligomeric state
Size exclusion can be used to determine oligomeric state

Vo = Void volume (the excluded volume surrounding the beads)

Ve = Intermediate volume (partially excluded)

Construct a standard curve using known proteins of known sizes

slide29
A protein’s substrate preference can be used

in a very specific purification step

Intrinsic

If a protein binds ATP, put over a column

support that has ATP crosslinked on it, thus

selecting for ATP-binding proteins (can be done

or a wide range of substrates such as sugars,

Proteins, etc.)

Added

Specific protein domains can be fused to proteins

of interest at the gene level to facilitate purification

(ie. Fuse a maltose binding protein domain to any

random protein, then it will bind specifically to a

maltose containing column)

slide30
Metal chelation is a popular affinity

purification method

Various “expression vectors” create fusions to

poly-Histidine tags, which allow the protein to bind to

columns containing chelated metal supports (ie. Ni+2)

Figures from Qiagen

Product literature

slide36
We can “control” protein expression

With the notable exception of proteins such as

those that compose the ribosome, many proteins

are found only in low abundance (particularly

Proteins involved in regulatory processes)

Thus, we need to find ways to grow cells that

allow ample expression of proteins that would

be interesting for biochemical characterization.

slide37
Find conditions for cell growth that enhance a

protein’s expression

For example, cytochrome c2 is utilized by R.sphaeroides

for both respiratory and photosynthetic growth; a slight

increase in levels of this protein is observed under

photosynthetic growth conditions.

However, Light-Harvesting complexes are only synthesized

under photosynthetic growth conditions; obviously if you

want to purify this protein you need to grow cells under

photosynthetic conditions

slide38
Molecular Biology allows us to manipulate genes
  • Understanding the basic mechanisms of gene expression
  • has allowed investigators to exploit various systems for
  • protein expression
  • Prokaryotic expression systems
  • Eukaryotic expression systems
  • Yeast
  • Mammalian
  • Viral expression systems

Baculovirus and Insects

slide39
Terminator

Promoter

lamB

Transcriptional unit

What do we need to produce a protein?

lamB

A gene

Ribosome binding site

lamB

Translational unit

slide40
Terminator

Promoter

lamB

Molecular Biology presents an opportunity for

useful genetic constructs

Antibiotic resistance gene

Origin of

Replication

ori

bla

Plasmid

Can fuse gene to other sequences conferring affinity

slide41
Choice of promoter allows control over

transcription levels

  • Intrinsic promoters can be sufficient for overexpression
  • in multi-copy plasmids
  • Constitutive promoters with high activity (ie. promoters for
  • ribosomal genes) can be useful for producing non-toxic
  • proteins
  • Inducible promoters allow control of expression, one can

“titrate” the promoter activity using exogenous agents

slide42
ori

bla

lamB

An expression system utilizing lactose and T7 RNA

polymerase is a popular choice in prokaryotes

Genome

Plasmid

T7 polymerase

dependent promoter

T7 pol

Lactose-inducible

promoter

slide43
Inclusion bodies provide a rapid purification step

Proteins exist

as aggregates in inclusion

bodies thus special

precautions must be taken

during purification. Typically,

inclusion bodies can be readily

isolated via cell fractionation.

following isolation the proteins

must be denatured and renatured

to retrieve active protein.

slide44
Additional concerns regarding protein expression

Modifications

Inclusion bodies

Codon usage

slide45
Cells exhibit nonrandom usage of codons

This provides a mechanism for regulation;

however, genes cloned for purposes of

heterologous protein expression may contain

“rare” codons that are not normally utilized by

cells such as E. coli. Thus, this could limit

protein production. Codon usage has been used

for determination of highly expressed proteins.

slide46
Molecular Biology allows us to manipulate genes
  • Understanding the basic mechanisms of gene expression
  • has allowed investigators to exploit various systems for
  • protein expression
  • Prokaryotic expression systems
  • Eukaryotic expression systems
  • Yeast
  • Mammalian
  • Viral expression systems

Baculovirus and Insects

slide47
Non-prokaryotic expression systems have emerged due to

increasing simplicity and the need for proper modifications.

Although you can express a eukaryotic cDNA in a prokaryote

is the protein you purify, what the eukaryotic cell uses?

Invitrogen : www.invitrogen.com

Gateway vectors

Novagen: www.novagen.com

slide48
Several hyperthermophilic archaeal species have also been shown

to be dependent on tungsten (W), also Cd important in diatoms

metals in biology
Metals in Biology
  • Enzyme co-factors

Redox active centers in many enzymes

Fe: Electron transport, SOD, Cytochrome P450

Zn: SOD

Mg, Mn: Photosynthesis

Cu: Electron transport

Ca: Cell signaling

Ca, Na, etc: Substrates in ion pumps

  • Structural components of enzymes

Fe: Hemoglobin, Cell structure

Zn: Zn fingers in transcription factors

Ca: Bone structure, Cell structure

metals and their biological effects
Metals and their biological effects
  • Block essential function of biomolecules

e.g. Ion pumps: Divalent metals inhibit Ca pumps

  • Displace essential metal co-factors

e.g. Cd can replace Cu in electron transport enzymes

  • Modify configuration of biomolecules: Zn can be replaced Cd in Zn fingers
metals and reactive oxygen species
Metals and reactive oxygen species
  • Redox potential of O2 ~ + 1 V; Extremely oxidizing
  • If there is a source of electrons:
  • O2 + e- O2- + e- + 2 H+ H2O2 + e-

 OH + OH- + e- + 2 H+ H2O

  • All but water are reactive oxygen species (ROS) and are biologically damaging
  • In above order: superoxide, hydrogen peroxide, hydroxyl radical
  • Biomolecules are a good source of reducing power: i.e. electrons
  • Redox active metals can catalyze electron transfer from biomolecules to O2
slide53
Metals, cannot be metabolized
  • Sequestered and/or excreted
  • Metallothioneins: Cu, Zn, Cd, Ni binding
  • Small sulphur containing proteins – free Cys residues
  • Bind to metals sequestering them

Cd++

S-

SH

+ Cd++

+ 2 H+

S-

SH

  • ~ 4 metal ions per protein
  • Binding region similar to Zn fingers
  • Expression induced by metal transcription factors (MTFs)
slide54
Metals in Enzymes
  • All ribozymes are metalloenzymes, divalent cations are required for
  • chemistry, and often aid in structural stabilization.
  • Protein enzymes are divided into six classes by the Enzyme Commision:
  • Oxidoreductase
  • Transferase
  • Hydrolase
  • Lyase
  • Isomerase
  • Ligase
  • Zn is the only element found in all of these classes of enzymes.
slide55
Proteins bind metals based on size, charge,

and chemical nature

Each metal has unique properties regarding ionic charge

ionic radii, and ionization potential

Typically, metals are classified as “hard” or “soft” in

correlation with their ionic radii, electrostatics, and

polarization

Hard metals prefer hard ligands, soft prefer soft,

Borderline metals can go either way.

slide57
Soft

Hard

slide58
L

L

L

L

L

M

M

L

L

L

L

L

L

L

M

M

L

L

L

L

L

L

L

Metals favor distinct coordination in proteins

Tetrahedral

Trigonal bipyramidal

M = Metal

L = Ligand

Octahedral

Square Planar

slide59
Unsaturated coordination spheres usually have water as

additional ligands to meet the favored 4 or 6 coordination

slide60
Protein sequence analyses have revealed certain metal

binding motifs

Structural Zn are generally bound by 4 cysteines

Catalytic Zn bound by three residues (H, D, E, or C) and one water

Coordination in primary sequence of alcohol dehydrogenase

Catalytic

L1-few aa-L2-several aa-L3

Structural

L1-3-L2-3-L3-8-L4

L = Ligand

slide62
Biological roles of transition metals

(not just limited to proteins*)

  • Coordination
  • Structure (protein and protein-substrate)
  • Electrophilic catalysis
  • Positive charge attracts electrons, polarize
  • potential reactant, increase reactivity
  • General Acid – Base catalysis
  • Redox reactions
  • Metalloorganic chemistry
  • Free radicals
slide64
Molybdenum??

http://www.dl.ac.uk/SRS/PX/bsl/scycle.html

slide65
Tetrapyrroles (heme, chlorophyll) make

proteins “visible” along with certain metals

slide66
Spectroscopy is a study of the interaction of

electromagnetic radiation with matter

A = ecl

Absorbance = extinction coefficient x concentration x path length

Units: None = M-1 cm-1 M cm

Beer-Lambert Law

The amount of light absorbed is proportional to the number of

molecules of the chromophore, through which the light passes

slide68
Purification of GFP overview
  • Protein stability
  • Protein precipitation
  • Hydrophobic Interaction chromatography
  • Gel electrophoresis
  • Optical spectroscopy
slide69
Lab reports

Introduction – Rationale for why these experiments

are important (not simply from a course work

perspective)

Materials & Methods – Concise, but detailed description

of how experiments were performed

Results – Summary of data (Simply report data, ie. purifica-

tion table, etc.)

Discussion – Implications of results

All lab reports must be type-written (please)

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