Part I- Protein PurificationEach protein folds into a specific shape and has a unique 3-D conformation.

Objectives of Protein Purification Step:• To separate one protein from a complex mixture of

proteins (such as a tissue extract), while maintaining Biological Function.

• One can maintain biological function by controlling the pH, the temperature and the ionic strength (ie. salt concentration).

Considerations in Protein PurificationA. Selection of Protein Source

• From natural source• From E. Coli host cells through molecular cloning• From Yeast host cells through molecular cloning• From mammalian cells or insect cells through transfection

B. Protein solubility and stability• obtain crude protein extracts from host cells• stabilization of proteins

* Native State: adjust pH, ionic strength, temp, buffer solution constituents, reducing reagent etc.* denature � renature

C. Assay of proteins• unique assay for protein of interest(For an enzyme, its biological activity is used as a unique assay for its presence in a tissue extract.)

• Assay for total protein and its’ purity.• functional assay

D. Quantity (How Much?), Quality (How Pure?)eg. Substance of < 0.1% tissue dry weight � 98% pure

Protein Purification Strategies

To purify the protein you use methods based on differences in:

1. Surface charge on proteins2. Molecular size of protein (related to molecular

weight)3. Biological activity due to binding to substrates or

inhibitors)4. Other chromatographic methods (reverse-phase,

hydrophobic interaction etc.)

Ion exchange chromatographyThe surface charge, or the net charge, of the protein at a set pH will be negative, neutral, or positive which depend on the pI of the protien. Thus, one can use ion exchange chromatography to separate proteins with differing charges.

Principles of ion exchange chromatography

Ion exchange chromatography (gradient)

Ion exchange chromatography

Gel Filtration ChromatographyAlso called Size exclusion chromatography, The size of protein is related to its’ molecular Weight. Thus, proteins can be separated like particles in a sieve-done with gel beads.

Gel Filtration Chromatography

Gel bead has molecular size holes so that small molecules like water and buffer enter it completely. Some proteins are small enough to also enter the molecular holes in the Gel bead. Other proteins are too large to enter holes and pass by the Gel bead.

In Gel Filtration, larger proteins elute first, medium sized ones next and finally smallest proteins elute last.

Standardization of gel filtration experiment

Vo: void volumeVe: the volume of solvent required to elute solute (protein etc) from the column.Ve/Vo: relative elution volume, which is independent of the size of the particular column used.

(1) Determine the molecular weight of the target molecule.

(2) Purification(3) Estimate the

polymerization state of the molecules.

Affinity ChromatographyAffinity chromatography depends on the biological activity or the affinity of the protein molecule for its substrates. It is usually a very effective method of purification. Sometimes, a protein can be purified to homogeneity (complete purity) using only affinity chromatography.

Affinity ChromatographyPurify monoclonal rat IgG using affinity chromatography

Lan1: rat hybridoma cell culture fluid, dilute 1;10Lane2: flow through, dilute 1:10Lane3: purified monoclonal rat IgG, dilute 1:5

Cross-linking of functional group

Extension of functional group

Coupling other groups

(1) spacer arm may be extended by reaction with succinic anhydride (VI)

(2) Phenolic group may be attached via diazonium derivatives(VII) or via bromoacetamidoalkyl derivative(V)

(3) Carboxyl group are coupled by carbodiimide reaction(III)

(4) Thiol derivative(VI) couple carboxyl groups in the presence of carbodiimide and the thiol ester bond may be cleaved using hydroxylamine.

Affinity Chromatography Application

Straviden + biotin

Streptavidin is a protein which acts like an antibiotic by removing biotin from the surrounding environment and inhibiting the growth of competitive microorganisms. The molecular weight of native Streptavidin is ~ 130,000 daltons(octameric protein), the molecular weight of the monomer is ~ 16,000 daltons. Streptavidin, like avidin, specifically binds the vitamin biotin. Unlike avidin, Streptavidin is not glycosylatedand shows significantly less non-specific binding. KD=10-15 M

Affinity Chromatography Application

DNA-binding protiensbinding site DNA

biotinylated nucleotide. mixture

tetrameric streptavidin ternarycomplex biotin-cellulose

Affinity Chromatography Application

GST(glutathio-S-transferase) catalyzes the addition of the glutathion sulfhydryl group to the epoxide, forming the first peptidoleukotrienes, leukotriene C4 (LTC4). Leukotrienes are involved in various inflammatory disorder and asthma.

�-glutamylcysteinylglycine,glutathione (GSH)

GST pull down purificationaffinity chromatography

” ”target protein

bait-protein: prepared as GST fusion proteinTest protein: either purified or labeled by in- vitro translation.

Metal Chelate Affinity ChromatographyMetGlySerSerHisHisHisHisHisHisSerSerGlyLeuValProArgGlySer....Example of a recombinant His-tag protein sequence

Hydrophobic Interaction Chromatography (HIC)Reverse Phase Chromatography (RPC)

The resin used in hydrophobic interaction method

-O-Si-CH2-CH2-CH2-CH3

CH3

CH3

-O-Si-(CH2)7-CH3

CH3

CH3

-O-Si-(CH2)17-CH3

CH3

CH3

C4 column

C8 column

C18 column

Agarose-octyl groupAgarose-Phenyl group

Reverse Phase Chromatography

DialysisPurpose:• Reduce ionic strength of the solution• Concentrate protein sample

SUMMARY of PURIFICATION PROCEDURES

To obtain a homogeneous protein/enzyme, one must apply a combination of the methods.

For example, ion-exchange chromatography is applied first, which separates proteins by difference in ionic properties;

Then gel filtration is applied, which separates by difference in molecular size. Since few proteins have exactly the same ionic character and same molecular size, a high degree of purity will be achieved by a combination of these two purification methods.

Affinity chromatography, which is much more specific for a particular enzyme, will in the end usually render an enzyme homogeneous.