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

Enzymatic: lysozyme, glucanases, chitinase

Physical: osmotic shock, freeze/thawMechanical: sonication, homogenization,

wet milling, French press

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

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

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

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

Differential centrifugation

Inclusion bodies provide a rapid purification step

Inclusion bodies providestorage space for protein,carbohydrate and lipid material in prokaryotes

However, proteins existas aggregates in inclusionbodies thus special precautions must be takenduring purification

10%

Glycerol

40%

Glycerol

Even proteins can be separated by their sedimentation properties

Function of both size and shape

Proteins have unique properties resulting from their amino acid composition

Localization

Charge

Hydrophobicity

Size

Affinity for ligandsArbitrary protein

The charge on a protein is dependent upon pH

The content of amino acids with ionizableside chains determines the overall charge of a protein

Thus, a protein containing a majority of basicresidues (ie. R and K) will be positively chargedand will bind to a cation-exchange support

Ion exchange columnSupports (examples)

Separation based on surface charge

Cation exchange chromatography

Protein- -

--- - -

-- - ----

- --

--

-Na+Cl-

Protein samples are applied tothis column at low ionic strength,and positively charged proteinsbind to the column support

Proteins are eluted using a gradientof increasing ionic strength, wherecounterions displace bound protein, changing pH will also elute protein

Choice of functional groups ondistinct column supports allow arange of affinities

Conversely, at a pH two orders of magnitude above their pKa, acidic amino acids will benegatively charged, thus proteins with a majority ofacidic amino acids (D and E) will be negativelycharged at physiological pH

Negatively charged proteins can be separated usinganion exchange chromatography

Na+Cl-Protein

Protein samples are applied tothis column at low ionic strength,and negatively charged proteinsbind to the column support

Proteins are eluted using a gradientof increasing ionic strength, wherecounterions displace bound protein, changing pH will also elute protein

Choice of functional groups ondistinct column supports allow arange of affinities

Bead size affects resolution in bothanion and cation exchange

Anion exchange chromatography

+

+

+ +

++ +++ + + +

+++

+ ++++

Hydrophobic Interaction Chromatography

Although most hydrophobic amino acids are buried inthe interior of proteins, many proteins have hydrophobicsurfaces or patches which can be used for separation

A protein’s hydrophobic character is typically enhancedby addition of high salt concentrations

Proteins are eluted from HIC columns via a gradient ofhigh salt to low salt concentrations

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 Focusing

Isoelectric focusing

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

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

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-

Salt effects on protein solubility

Salting out provides a purification step

Proteins can be separated on the basis of size

Gradient centrifugation

Gel filtration

Gel Filtration provides a molecular sieve

Figures from Scopes, Protein Purificationon Reserve

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

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

Ve = Intermediate volume (partially excluded)

Construct a standard curve using known proteins of known sizes

Gel Filtration Chromatography

LogMolWt

Ve - Vo

A protein’s substrate preference can be used in a very specific purification step

IntrinsicIf a protein binds ATP, put over a columnsupport that has ATP crosslinked on it, thusselecting for ATP-binding proteins (can be doneor a wide range of substrates such as sugars, Proteins, etc.)

AddedSpecific protein domains can be fused to proteinsof 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 amaltose containing column)

Metal chelation is a popular affinity purification method

Various “expression vectors” create fusions topoly-Histidine tags, which allow the protein to bind tocolumns containing chelated metal supports (ie. Ni+2)

Figures from QiagenProduct literature

Examining your purified protein

Use of SDS-PAGE vs. Native gel electrophoresis

Two dimensional gel electrophoresis

Assessing your purification procedure

Total vs. specific activity

We can “control” protein expression

With the notable exception of proteins such asthose that compose the ribosome, many proteinsare found only in low abundance (particularlyProteins involved in regulatory processes)

Thus, we need to find ways to grow cells thatallow ample expression of proteins that would be interesting for biochemical characterization.

Find conditions for cell growth that enhance a protein’s expression

For example, cytochrome c2 is utilized by R.sphaeroidesfor 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 synthesizedunder photosynthetic growth conditions; obviously if you want to purify this protein you need to grow cells underphotosynthetic conditions

Molecular Biology allows us to manipulate genes

Understanding the basic mechanisms of gene expressionhas allowed investigators to exploit various systems for protein expression

Prokaryotic expression systems

Eukaryotic expression systemsYeastMammalian

Viral expression systemsBaculovirus and Insects

What do we need to produce a protein?

lamB

A gene

lamB

Promoter

Transcriptional unit

Terminator

lamB

Ribosome binding site

Translational unit

Molecular Biology presents an opportunity for useful genetic constructs

lamB

Promoter Terminator

bla

Plasmid

oriAntibiotic resistance gene Origin ofReplication

Can fuse gene to other sequences conferring affinity

Choice of promoter allows control over transcription levels

Intrinsic promoters can be sufficient for overexpressionin multi-copy plasmids

Constitutive promoters with high activity (ie. promoters forribosomal genes) can be useful for producing non-toxicproteins

Inducible promoters allow control of expression, one can “titrate” the promoter activity using exogenous agents

An expression system utilizing lactose and T7 RNA polymerase is a popular choice in prokaryotes

lamB

blaori

Plasmid

T7 polymerasedependent promoter

T7 polLactose-inducible promoter

Genome

Inclusion bodies provide a rapid purification step

Proteins existas aggregates in inclusionbodies thus special precautions must be takenduring purification. Typically,inclusion bodies can be readilyisolated via cell fractionation.following isolation the proteinsmust be denatured and renaturedto retrieve active protein.

Additional concerns regarding protein expression

Modifications

Inclusion bodies

Codon usage

Cells exhibit nonrandom usage of codons

This provides a mechanism for regulation;however, genes cloned for purposes ofheterologous protein expression may contain“rare” codons that are not normally utilized bycells such as E. coli. Thus, this could limit protein production. Codon usage has been usedfor determination of highly expressed proteins.

Molecular Biology allows us to manipulate genes

Understanding the basic mechanisms of gene expressionhas allowed investigators to exploit various systems for protein expression

Prokaryotic expression systems

Eukaryotic expression systemsYeastMammalian

Viral expression systemsBaculovirus and Insects

Non-prokaryotic expression systems have emerged due toincreasing 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

Several hyperthermophilic archaeal species have also been shown to be dependent on tungsten (W), also Cd important in diatoms

Fe is most abundant, followed by Zn

Metals in Biology

• Enzyme co-factorsRedox active centers in many enzymesFe: Electron transport, SOD, Cytochrome P450Zn: SODMg, Mn: PhotosynthesisCu: Electron transportCa: Cell signalingCa, Na, etc: Substrates in ion pumps

• Structural components of enzymes Fe: Hemoglobin, Cell structureZn: Zn fingers in transcription factorsCa: Bone structure, Cell structure

Metals and their biological effects

• Block essential function of biomolecules e.g. Ion pumps: Divalent metals inhibit Ca pumps

• Displace essential metal co-factorse.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

• 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

• 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

SHSH + Cd++

S-

S- + 2 H+Cd++

• ~ 4 metal ions per protein

• Binding region similar to Zn fingers

• Expression induced by metal transcription factors (MTFs)

Metals in Enzymes

All ribozymes are metalloenzymes, divalent cations are required forchemistry, and often aid in structural stabilization.

Protein enzymes are divided into six classes by the Enzyme Commision:1. Oxidoreductase2. Transferase3. Hydrolase4. Lyase5. Isomerase6. Ligase

Zn is the only element found in all of these classes of enzymes.

Proteins bind metals based on size, charge, and chemical nature

Each metal has unique properties regarding ionic chargeionic radii, and ionization potential

Typically, metals are classified as “hard” or “soft” incorrelation with their ionic radii, electrostatics, andpolarization

Hard metals prefer hard ligands, soft prefer soft,Borderline metals can go either way.

Properties of metal ions determine their biological utility

Soft

Hard

Metals favor distinct coordination in proteins

M

LL

L L

M

L L

LLM

L

LL

LL

LM

L

L L

L

L

Tetrahedral

Square Planar

Trigonal bipyramidal

Octahedral

M = MetalL = Ligand

Unsaturated coordination spheres usually have water as additional ligands to meet the favored 4 or 6 coordination

Protein sequence analyses have revealed certain metalbinding 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

CatalyticL1-few aa-L2-several aa-L3

StructuralL1-3-L2-3-L3-8-L4

L = Ligand

Biological roles of transition metals

Coordination Structure (protein and protein-substrate)Electrophilic catalysis Positive charge attracts electrons, polarize potential reactant, increase reactivityGeneral Acid – Base catalysisRedox reactionsMetalloorganic chemistry Free radicals

(not just limited to proteins*)

Carbonic Anhydrase catalytic mechanism

Molybdenum??

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

Tetrapyrroles (heme, chlorophyll) makeproteins “visible” along with certain metals

Spectroscopy is a study of the interaction of electromagnetic radiation with matter

A = cl

Absorbance = extinction coefficient x concentration x path length

Beer-Lambert Law

The amount of light absorbed is proportional to the number ofmolecules of the chromophore, through which the light passes

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

c-type cytochromes have a characteristic absorbance spectrum

Purification of GFP overview

Protein stability

Protein precipitation

Hydrophobic Interaction chromatography

Gel electrophoresis

Optical spectroscopy

Lab reports

Introduction – Rationale for why these experimentsare important (not simply from a course workperspective)

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)

Keeping a purification table