Protein Purification

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Protein Purification
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Compartmentalization provides an opportunity
for a purification step
e
Protein profile for compartments of gram-negative
prokaryotes
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Cell Disruption

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

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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

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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.

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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

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Sonication
A sonicator can be immersed directly into a cell
suspension. The sonicator is vibrated and high
frequency sound waves disrupt cells.
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Differential centrifugation
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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
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Even proteins can be separated by their
sedimentation properties
Function of both size and shape
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Proteins have unique properties resulting
from their amino acid composition

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

Arbitrary protein
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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)
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Separation based on surface charge
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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

NaCl-
Protein
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• 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

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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

NaCl-
Protein




















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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 proteins 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

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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.

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Isoelectric focusing
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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

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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

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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 gt K gt Na gt Mg gt
Ca gt guanidium Anions SO4-- gt HPO4-- gt
acetate gt citrate gt tartrate gt Cl- gt NO3-
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Salting out provides a purification step
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Proteins can be separated on the basis of size

• Gradient centrifugation
• Gel filtration

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Gel Filtration provides a molecular sieve
Figures from Scopes, Protein Purification on
Reserve
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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

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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
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Gel Filtration Chromatography
Log Mol Wt
Ve - Vo
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A proteins 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)
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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. Ni2)
Figures from Qiagen Product literature
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Examining your purified protein
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Use of SDS-PAGE vs. Native gel electrophoresis
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Two dimensional gel electrophoresis
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Assessing your purification procedure
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Total vs. specific activity
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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.
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Find conditions for cell growth that enhance a
proteins 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 photosyntheti
c conditions
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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

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What do we need to produce a protein?
lamB
A gene
Ribosome binding site
lamB
Translational unit
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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
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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

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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
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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.
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Additional concerns regarding protein expression
Modifications Inclusion bodies Codon usage
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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.
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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

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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
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Several hyperthermophilic archaeal species have
also been shown to be dependent on tungsten (W),
also Cd important in diatoms
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Fe is most abundant, followed by Zn
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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

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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

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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

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• 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)

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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.

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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.
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Properties of metal ions determine their
biological utility
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Soft
Hard
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Metals favor distinct coordination in proteins
Tetrahedral
Trigonal bipyramidal
M Metal L Ligand
Octahedral
Square Planar
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Unsaturated coordination spheres usually have
water as additional ligands to meet the favored
4 or 6 coordination
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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
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(No Transcript)
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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

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Carbonic Anhydrase catalytic mechanism
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Molybdenum??
http//www.dl.ac.uk/SRS/PX/bsl/scycle.html
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Tetrapyrroles (heme, chlorophyll) make proteins
visible along with certain metals
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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
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c-type cytochromes have a characteristic
absorbance spectrum
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Purification of GFP overview

• Protein stability
• Protein precipitation
• Hydrophobic Interaction chromatography
• Gel electrophoresis
• Optical spectroscopy

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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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Keeping a purification table