Protein purification
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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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Protein Purification

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Protein purification

Protein Purification


Protein purification

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.


Differential centrifugation

Differential centrifugation


Protein purification

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


Protein purification

10%

Glycerol

40%

Glycerol

Even proteins can be separated by their

sedimentation properties

Function of both

size and shape


Protein purification

Proteins have unique properties resulting

from their amino acid composition

  • Localization

  • Charge

  • Hydrophobicity

  • Size

  • Affinity for ligands

Arbitrary protein


Protein purification

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)


Separation based on surface charge

Separation based on surface charge


Protein purification

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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Protein purification

  • 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


Protein purification

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

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Protein purification

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.


Isoelectric focusing1

Isoelectric focusing


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


Protein purification

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-


Protein purification

Salting out provides a purification step


Protein purification

Proteins can be separated on the basis of size

  • Gradient centrifugation

  • Gel filtration


Protein purification

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


Protein purification

Gel Filtration Chromatography

Log

Mol

Wt

Ve - Vo


Protein purification

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)


Protein purification

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


Examining your purified protein

Examining your purified protein


Use of sds page vs native gel electrophoresis

Use of SDS-PAGE vs. Native gel electrophoresis


Two dimensional gel electrophoresis

Two dimensional gel electrophoresis


Assessing your purification procedure

Assessing your purification procedure


Total vs specific activity

Total vs. specific activity


Protein purification

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.


Protein purification

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


Protein purification

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


Protein purification

Terminator

Promoter

lamB

Transcriptional unit

What do we need to produce a protein?

lamB

A gene

Ribosome binding site

lamB

Translational unit


Protein purification

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


Protein purification

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


Protein purification

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


Protein purification

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.


Protein purification

Additional concerns regarding protein expression

Modifications

Inclusion bodies

Codon usage


Protein purification

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.


Protein purification

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


Protein purification

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


Protein purification

Several hyperthermophilic archaeal species have also been shown

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


Protein purification

Fe is most abundant, followed by Zn


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


Protein purification

  • 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)


Protein purification

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.


Protein purification

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.


Protein purification

Properties of metal ions determine their biological utility


Protein purification

Soft

Hard


Protein purification

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


Protein purification

Unsaturated coordination spheres usually have water as

additional ligands to meet the favored 4 or 6 coordination


Protein purification

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


Protein purification

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


Protein purification

Carbonic Anhydrase catalytic mechanism


Protein purification

Molybdenum??

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


Protein purification

Tetrapyrroles (heme, chlorophyll) make

proteins “visible” along with certain metals


Protein purification

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


Protein purification

c-type cytochromes have a characteristic

absorbance spectrum


Protein purification

Purification of GFP overview

  • Protein stability

  • Protein precipitation

  • Hydrophobic Interaction chromatography

  • Gel electrophoresis

  • Optical spectroscopy


Protein purification

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)


Protein purification

Keeping a purification table


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