Other Protein Purification Methods

What is the purpose of protein purification

• Many experiments require pure protein solution

  • Essential for studying structure and function

• Protein isolation methods take advantage of the characteristics of proteins

  • Size

  • Charge

  • Solubility

  • Binding properties

What characteristics of proteins are used in protein purification

• Size

• Charge

• Solubility

  • i.e. Ammonium Sulphate precipitation

• Binding properties

  • Best protein characteristic to take advantage of

  • Need to know characteristic of protein to use

    • Less specific: Several proteins in the sample could share similar properties

    • More specific: Specific proteins specifically bind other molecules/ligands

What are the tools used for protein purification

• Salt Fractionation (we utilized this method for RuBisCo isolation)

• Chromatographic assays

  • Take advantage of adsorption

    • Size-exclusion chromatography

    • Ion-exchange chromatography

    • Affinity chromatography

      • Tagging proteins

Define adsorption and adsorbent

Adsorption = The process by which ions, atoms or molecules adhere to the surface of a solid material

• Note: Different to absorption; adsorption enters into a liquid absorbent

• Used in chromatography

• Intramolecular forces (ionic, hydrogen bonds, Van der Waals) causes adhesion

Adsorbent = a substance which is generally porous in nature with a high surface area to adsorb substances on its surface by intermolecular forces

What is the purpose of chromatography

Chromatography is used to separate individual compounds from a mixture

• Takes advantage of adsorption

What are the stationary and mobile phases of chromatography

Stationary phase = a column of adsorbent

• Usually on top

Mobile phase = A solvent (liquid or gas) moves through mixture of different compounds through the stationary phase

• Usually a buffer where you dissolve the mixture of solvent

• Usually on the bottom

What is column chromatography

• Porous solid material (stationary phase)

• Buffered solution (mobile phase)

• Mixture separation:

  • Mobile phase moves through stationary phase, thus carrying the solutes/components of mixture

  • Components travel down the column and take position on column based on affinity towards adsorbent

    • Strongly adsorbed compounds seen at top
      of column

What is size-exclusion chromatography

Used when you know the molecular weight of the protein of interest

• Mobile phase = Buffer

• Stationary phase = Cross-linked polymer beads with pores of specific sizes

• Depending on the pore and protein size, proteins can enter or not the pores and get retained by the stationary phase

• Separates proteins by size

  • Large proteins emerge first

what is ion-exchange chromatography and how does charge affect elution speed

Used when you know the specific charge of a protein at a certain pH

• Separates proteins based on differences in their net electrical charge

• Mobile phase = Buffer at specific pH

• Stationary phase = Porous solid synthetic resin (beads) coated with charged groups

• Anionic groups (-) = cation exchange

• Bind to and slows positively charged protein

• Cationic groups (+) = anion exchange

• Bind to and slows down negatively charged protein

In a Cation Exchange Column (resin negatively charged)

• Proteins with a large net positive charge at the specific column pH get retained more efficiently by the stationary phase and elute slower

• Proteins with a large net negative charge at the specific column pH get repelled by the negative charges in the stationary phase and elute faster

  • The opposite occurs in an Anion Exchange column (resin positively charged)

what is affinity chromatography

• Based on binding affinity to a specific ligands

• We need to know the ligand the target protein specifically binds

  • Stationary phase: Porous solid with covalently attached ligand

  • Specific protein will bind specific ligands

• Elution with excess of soluble ligand

Define FPLC

Fast Protein Liquid Chromatography

• FPLC is based on ion exchange

• Uses pressure from pumps to control speed at which the mobile phase passes through stationary phase

  • System pumps get the buffers on the column

  • Sample pump gets protein solution on the column

What factors determine the net charge of a protein

Depends on:

• Amino acid composition

• pH

By changing the pH of the mobile phase, you can change the charge of the proteins in the solution

How is the equilibrium of acids and bases defined

Acid dissociation constant (K_a) = Describes the extent of dissociation of weak acid in solution at equilibrium

• K_a = [A^-][H⁺] / [HA]

or pK_a

• pK_a = log₁₀ [HA] / [A^-][H⁺] = —log₁₀K_a

How can the equilibrium of acids and bases be shifted

As the equilibrium depends on the concentration of the reacting species,

• We can shift the equilibrium by changing the concentration of one of them

  • i.e. the concentration of protons → pH

When the pH of the solution gets lower than the pK_a of the compound, the compound will exist predominately in its protonated form (HA)

What is the Henderson-Hasselbach equation

pH - pK_a = log [A^-] / [HA] for weak acids

• if pH < pK_a = protonated form (HA) predominates (A^- / HA ratio < 1)

  • Will be negative

  • [A^-] is lower than [HA]

• if pH > pK_a = deprotonated form (A^-) predominates

  • Will be positive

  • [A^-] is higher than [HA]

Define the isoelectric point (pl). How is pl calculated and which amino acids of a protein will predominantly determine the pl

Isoelectric point of Amino Acids = pH at which a molecule carries no net electrical charge (net charge = 0)

• pI = pK₁ + pK₂ / 2

• If pH < pK = molecule/group becomes protonated

• If pH > pK = molecule/group becomes deprotonated

• In proteins, only 1 amino and 1 carboxyl group can be ionized in the peptide backbone (N and C terminus), influencing the pI

• However,

  • Ionizable side chains in the R groups can influence pI

  • Posttranslational modification can introduce new ionizable groups and affect pI

  • Amino acid individual pI may change when in a peptide

What if you want your protein to be positively or negatively charged, not neutral

In a buffered solution where pH is below proteins pI → the protein is (+)
charged (protonated):

• will bind (-) charged functional groups of a cation exchange resin

In a buffered solution where pH is above proteins pI, the protein is (-)
charged (deprotonated):

• will bind (+) charged functional groups of anion exchange resin

• Different proteins have different pIs

  • At the same pH, different proteins will have different charges

what are the events that occur during equilibration, sample application, elution, and regeneration steps of the ion-exchange chromatography

1) Equilibration

• Add a buffer containing ions into column

• negatively charged ions of buffer interact w/ positively charged ionic groups of beads.

2) Sample application

• Protein gets adsorbed in the resin

• Negatively Charged proteins attach to resin/beads

  • Same buffer as #1

3) Elution

• means to remove from the stationary stage

• Recovering the protein of interest

  • Protein can be eluted by changing pH

  • or by increasing salt concentration/ionic strength

4) Regeneration

• Wash with high ionic strength buffer to strip off remaining protein from column

What is the purpose of tagging the protein of interest and what is its advantages and disadvantages

Tagging a protein allows us to bind it to something when we don’t know the specific ligand it binds to

• Many proteins do not bind to a convenient ligand

• We can modify the gene for the target protein to add a ligand binding site-TAG

  • Fusion protein; gene for a tag protein is placed next to the gene for the target protein

  • The tag and target protein will be translated and expressed together

• Advantage: Allows affinity chromatography when we don’t the specific ligand that the protein of interest binds to

• Disadvantage: may interfere with protein function

What are some examples of ligand-binding TAGs and their corresponding ligand

• His - tag in Affinity Chromatography

  • Tag of 6 or more histidine residues that make proteins bind immobilized Ni²⁺ ions

    • Tag protein/peptide = (His)₆

    • Ligand = Ni²⁺

• Immobilized protein A (and G)

  • For non-specific purification of antibodies

  • Produced by bacteria as defense mechanism

  • Matrix bound protein A strongly binds the Fc region of immunoglobulin G (IgG)

  • Fc region of all IgG antibodies is the same (for each species)

    • Tag protein/peptide = Protein A or G

    • Ligand = Fc portion of IgG

• Immobilized antigens

  • for specific purification of antibodies

  • Specific antigens are immobilized in the chromatographic column

  • Mixture of antibodies gets loaded in the column.

  • The antibodies bind the immobilized antigens depending on the specificity of their Fab region for the antigen

  • Antibodies get eluted

    • Tag protein/peptide = Glutathione-S-transferase (GST)

    • Ligand = Glutathione