Protein purification is a multi-step process

Macromolecular composition of E. coli strain B/r grown under a standard culturecondition (i.e., balanced growth, glucose minimal medium, 37°C, mass doubling time of 40 min.):

Macromolecules:

Protein 55% (of total dry weight)

RNA 20.5%

DNA 3.1%

Lipid 9.1%

Lipopolysaccharide 3.4%

Murein 2.5%

Glycogen 2.5%

Soluble pool

(amino acids,

vitamins, etc.) 2.9%

Inorganic ions 1.0%

Estimate 1 g of dry weight cellsper 1 L growth medium, thisyields ~550 mg of protein

Some proteins are abundant (ie.ribosomal proteins) while otherscan represent 0.1% of this total

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)

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

+

+

+ +

++ +++ + + +

+++

+ ++++

At a specific pH

, the isoelectric point, a protein w

ill not exhibit a charge

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

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

10%

Glycerol

40%

Glycerol

Proteins can be separated by their sedimentation properties

Function of both size and shape

Gel Filtration provides a molecular sieve

Figures from Scopes, Protein Purificationon Reserve

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

Carbonic Anhydrase is a non-abundant protein in M. thermophila

Increasing purity

Taken from paper on reserve

Purification Analysis

Spectroscopy

Gel Electrophoresis

Enzymatic Assay

Why is the sky blue?

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

How do molecules absorb light?

Photon energy causes electrons to jump to higher energy levels in molecular orbitals

Increased delocalization shifts the absorption band closer to the visible range

Tetrapyrroles (heme, chlorophyll) make proteins visible

c-type cytochromes have a characteristic absorbance spectrum

Isobestic point

About one-third of all enzymes require one or more metal ions for catalytic activity

Some transition metals absorb in the visible range

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

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.

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*)

*why did we add EDTA to lyse the outer membrane

Chelators bind metals tightly via multiple interactions

These may be difficult to make out, to get the idea simply look atthe heme figure

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

Transition metals are Lewis acids

Lowry-Bronsted – an acid is a substance that gives up a proton, and a base accepts a proton

Lewis – an acid can take up an electron pair to form a covalent bond, while a base can furnish an electron pair

Acid-Base definitions

Zinc acts as a Lewis acid to generate nucleophiles Nucleophiles are Lewis bases

Zn promotes formation of a nucleophile

Protein’s migrate as a function of size using SDS-PAGE

Molecular Weight Markers

Purified 20SProteasome

subunitsubunit

SDS is a strongly anionic detergentthat associates with denaturedpolypeptides proportionally to theirsize, thus the charge on a proteinbecomes independent of it’s sequencebut a function of it’s size, however, the sample must be completely denatured by heat and reductant prior to loading

Good analysis for an “apparent”molecular weight, and subunitcomposition

Protein’s migrate as a function of charge and size under non-denaturing conditions

Non-denaturing conditions allow you to examinethe native active state of an enzyme (can stain proteinsbased on their activity)

How many bands would you observe on a non-denaturing gel using purified proteasome?

How would you determine the stoichiometry of oligomeric enzymes using this technique?

Gel electrophoresis can be used in preparative as well as analytical ways

Gel electrophoresis can also separate proteins by pH (Isoelectric focusing)

High pH Low pH

pI =7.5

pI=6.8

pI=5.4

+-

Anti-sera can be used to detect specific proteins

Sufficiently separated proteins in an SDS-PAGE can be transferred to a solid membrane for Western Blot analysis. For this procedure, an electric current is applied to the gel so that the separated proteins transfer through the gel and onto the membrane in the same pattern as they separate on the SDS-PAGE. All sites on the membrane which do not contain blotted protein from the gel can then be non-specifically "blocked" so that antibody (serum) will not non-specifically bind to them, causing a false positive result.

To detect antigen blotted on the membrane, a primary antibody (serum) is added at an appropriate dilution and incubated with the membrane. If there are any antibodies present which are directed against one or more of the blotted antigens, those antibodies will bind to the protein(s) while other antibodies will be washed away at the end of the incubation. In order to detect the antibodies which have bound, anti-immunoglobulin antibodies coupled to a reporter group such as the enzyme alkaline phosphatase are added (e.g. Goat anti-human IgG- alkaline phosphatase). This anti-Ig-enzyme is commonly called a "second antibody" or "conjugate". Finally after excess second antibody is washed free of the blot, substrate is added which will precipitate upon reaction with the conjugate resulting in a visible band where the primary antibody bound to the protein.

Developing a Western Blot

Enzymatic assays measure enzyme activity

Time

Cha

nge

in s

omet

hing

(

Usu

ally

O. D

.)

Specific Activity vs. Total Activity