Transcript Protein Purification

Protein Purification
What is protein purification?
Protein purification is the separation of a specific protein from
contaminants in a manner that produces a useful end product.
Why purify proteins?
In a research environment, proteins must be purified in order to
determine their structure and study their biochemical properties.
In industrial settings, proteins are purified on a larger scale in order to be
sold as products such as drugs, vaccines, diagnostic tools or food
additives.
Issues: purity, yield and cost
Separation methods are based on protein properties. Because of
the differences in amino acid composition and sequence, and the
possible presence of non-protein groups, each protein has different
chemical characteristics that make it unique.
These characteristics include
 size (molecular weight)
 Charge
 solubility
 hydrophobicity
 biological affinity
Differences in these characteristics are the basis for separation
methods such as
 Filtration
 salt precipitation
 chromatographic procedures
Protein could be
 included inside the cell or
 secreted out of the cell.
Depending on which you will need to isolate the cells or the media.
Generally achieved by centrifugation or possibly filtration.
If the protein is within the cells the next step needed is lysis of cells.
Techniques used for the physical disruption of cells.
Lysis
Method
Description
Apparatus
Mechanical
Waring Blender
Polytron
Rotating blades grind and disperse cells and
tissues
Liquid
Homogeni
zation
Homogenizer
French Press
Cell or tissue suspensions are sheared by forcing
them through a narrow space
Sonication
Sonicator
High frequency sound waves shear cells
Freeze/Thaw
Freezer or dry
ice/ethanol
Repeated cycles of freezing and thawing disrupt
cells through ice crystal formation
Manual
grinding
Mortar and pestle
Grinding plant tissue, frozen in liquid nitrogen
Lysis methods cont.
2. Chemical methods
-chloroform or toluene-to solubilize the membrane, alkaline NaOH,
detergent, extreme salt concentrations
3. Enzymatic methods
-Lysozyme with or without EDTA to digest the
peptidoglycan layer
Next the protein needs to be separated either from the contents of the
spent media or from the contents of the lysed cell.
A variety of methods are used to separate out the protein ,
including some of the following:
1. Filtration
• In Ultrafiltration, molecules such as proteins and nucleic
acids are retained by the filter.
• These filters can only separate very large proteins from
very small proteins; they are mainly used for
concentrating proteins and for exchanging buffers.
2. Protein Precipitation
This step is used at an early step on crude material.
• A protein precipitate will form when proteins are
prevented from interacting with the surrounding water
molecules e.g. "Salting Out": Salts such as ammonium
sulfate., changing pH, heat denaturation
3. Ion exchange chromatography
• Proteins are made up of twenty common amino
acids.
• Some of these amino acids possess side groups
("R" groups) which are either positively or
negatively charged.
• A comparison of the overall number of positive and
negative charges will give a clue as to the nature of
the protein.
• If the protein has more positive charges than
negative charges, it is said to be a basic protein.
• If the negative charges are greater than the positive
charges, the protein is acidic.
• When the protein contains a predominance of ionic
charges, it can be bound to a support that carries
the opposite charge.
• A basic protein, which is positively charged, will
bind to a support which is negatively charged.
• An acidic protein, which is negatively charged,
will bind to a positive support.
• The use of ion-exchange chromatography, then,
allows molecules to be separated based upon
their charge.
• Families of molecules (acidics, basics and
neutrals) can be easily separated by this
technique.
• A very frequently used chromatographic
technique for protein purification.
• Matrix example DEAE
3. ion-exchange chromatography
• allows molecules to be separated based
upon their charge
4. Hydrophobic Interaction Chromatography
• Not all of the common amino acids found in
proteins are charged molecules.
• some amino acids that contain hydrocarbon
side-chains which are not charged
• cannot be purified by ion-exchange
chromatography.
• These hydrophobic amino acids are usually
buried away in the inside of the protein as it folds
into it's biologically active conformation.
• Some distribution of these hydrophobic residues
on the surface of the molecule.
• These hydrophobic amino acids can bind on a support
which contains immobilized hydrophobic groups.
• HIC supports work by a "clustering" effect; no covalent or
ionic bonds are formed or shared when these molecules
associate.
• Hydrophobic residues are alanine, valine, leucine,
isoleucine, phenylalanine, tryptophan, methionine,
proline.
• High salt exposes the hydrophobic groups which then
bind to the matrix.
• Elution is carried out by lowering the salt concentration
or with organic solvents if necessary.
• Matrix examples: phenyl or octyl sepharose
4. Hydrophobic Interaction Chromatography
5. Gel-Filtration Chromatography
• separates proteins based on size and shape.
• The support for gel-filtration chromatography are beads
which contain holes, called "pores," of given sizes.
• Larger molecules, which can't penetrate the pores, move
around the beads and migrate through the spaces which
separate the beads faster than the smaller molecules,
which may penetrate the pores.
• The matrix pore size determines the rate at which
different proteins can diffuse, and some proteins will be
completely excluded.
• The pore size chosen will
Depend on the specific
protein to be purified.
• The column is eluted with
buffer and the proteins come out
in order of the largest first.
• Matrix example: Sephadex
6. Affinity Chromatography
• It is the only technique which can potentially allow a one-step purification
of the target molecule.
• In order to work, a specific ligand (a molecule which recognizes the target
protein) must be immobilized on a support in such a way that allows it to
bind to the target molecule.
• E.g. the use of an immobilized protein to capture it's receptor (the reverse
would also work).
• Can be used for the purification of any protein, provided that a specific
ligand is available.
Ligand examples:
• a. Lectins such as wheat germ agglutinin or ConA which bind to the
carbohydrate portion of proteins
• b. Protein A or G which binds the Fc regions of some immunoglobulins
• c. Metal chelate chromatography — Zn columns bind histidine tagged
proteins.
• d. Dyes such as blue dextran