Lecture 7: Protein Purification and Identification

How to collect protein samples

• four important consideration

• Choosing the correct cell type or tissue is important for obtaining an adequate quantity and quality of sample.

• Important considerations:

  • Protein folding

  • Post-translational modifications (PTM)

  • Protein-protein interactions (separating to prevent conformational change)

  • Co-factors

• Molecular biology can help

  • Genetically engineer useful tags or sequences into your POI (to make the protein easier to find or grown).

  • Express your POI in a specific cell type or organism (e.g. E. coli).

Fusion proteins

• what is this process about?

• Why is it useful (possible additions to the proteins (5))

• Fuse protein domains together using molecular biology to purify and obtain the POI.

  • Fully functional protein or a small amino acid sequence called a protein tag (gene).

  • E.g. His tag (6-10 AA)

  • E.g. FLAG tag (positively and charged AA, binding of anti-FLAG to pull out of solution)

  • E.g. green fluorescent protein: for visualization (GFP)

• Adds useful features to proteins being studied

  • Fluorescence for visualization

    • E.g green fluorescent protein

  • Tags for affinity purification

    • Obtaining proteins from other non POI

  • Tags for Western blot detection

    • Antibodies for different tags

  • Enzymes for activity assays

    • May catalyze certain reaction to indicate the protein is present

  • Fused complexes for crystallization

    • Looking at the overall structure

Example picture

• Dark grey: the POI

• N-terminu: flag tag

• C-terminus: 6 histine rage (light gret)

• Thiroedoxin: help with solubility

• After purification, an exonuclease will cleave off the specific sites to remove the fusion proteins

Cell lysis

• what is it

• What cells is involved and what do they generate

• Three common methods to do this in a lab

• Two things to consider before doing it

• Lysing cells generates a crude extract.

  • Heterogenous mixture of proteins and cellular contents

  • To be able to obtain the proteins from within the cells

• Common Methods:

  • Mechanical/physical methods:

    • Grinding (peddle), Sonication (high frequency), Vortexing with glass beads (break apart membranes)

  • Osmotic Pressure (<20 mM NaCl)

    • Water to burst the cells

  • Chemical basis (e.g. detergents)

    • E.g. Detergents

• Important considerations:

  • Lysing cells may release proteases – enzymes (designed to break down unwanted proteins, and maybe your POI) )that may degrade your protein of interest (POI).

    • Solution? Protease inhibitors (ability to bind to the proteases and prevent functionality to break down your POI)

  • Conditions (pH/temp/detergents) may alter your protein’s structure and lead to denaturation.

    • Solution? Buffers + Ice (for pH issues)

    • Regulate the temp

Importance of protein purification

• there are many proteins in a mammalian cell (about 1 to 3 billion)

• Must isolate the particular protein from other cellular component in order to study it - very important when doing in vitro (inside a test tube rather than studying the organism as a whole)

  • A heterogenous population will contain other things that may affect the protein of interest

• Purifying a protein is a must step for understanding the protein structure/function

Centrifugation purpose

• what is the main purpose of a centrifugation

• Used to separate the supernatant (your POI including anything that is light) of soluble materials from a pellet of other large organelles or insoluble precipitate

  • Depend on what you study, you would either take the supernatant or pellet

• Allows for the isolation of a particular organelle

• Centrifugation can be combined with filters to separate by size

Chromatography

• what is the purpose of chromatography

• Chromatography: Differential partitioning of a molecule between a mobile (buffer) and stationary (resin; column) phase.

  • E.g. resin or beads for the stationary phase

• Proteins can be purified based on differences in their chemical properties:

  • Size or shape: Size-Exclusion/Gel Filtration chromatography

  • Charge: Ion Exchange Chromatography

  • Binding interactions: Affinity chromatography

  • Hydrophobicity: RP-HPLC (Reverse Phase High Pressure Liquid Chromatography)

Size exclusion chromatography

• Proteins are separated based on size and shape

• Columns contain a resin of porous beads

  • Smaller proteins enter the beads and move through the resin slowly (e.g. red beads) (more tubes used and came out later than the larger molecules)

  • Larger proteins bypass the beads and move through the resin quickly (e.g. yellow molecules) (more than 100 KDa = passes through)

  • Wait for timing, when your POI comes out or if they are completed excluded (depending on the size of the POI)

• Considerations:

  • Small but elongated proteins may appear larger – they may not enter the beads and elute faster

  • Calibration with proteins of known MW is required

  • MW = Molecular Weight

• Vo = void volume

  • Anything larger than the column’s fractional range (i.e. what doesn’t fit in the pores) goes straight through - bigger molecules

• Ve = elution volume of a molecule - the smaller molecules

• Vt = total volume of the column

• Issue: all molecules are colourless

  • Use UV absorbance to see when the POI comes out

Ion-exchange chromatography

• Separates molecules based on their net charge

• Recall the isoelectric point (pI) is the pH when a polypeptide is neutral

• Cation exchange resins attract and bind positively charged polypeptides

• Anion exchange resins attract and bind negatively charged polypeptides

• Proteins can be eluted by increasing the salt concentration or changing the pH

• Movement

  • Negatively charged or neutral go through the resin (column)

  • Positively charged does not go through

Affinity Chromatography

• Proteins are attracted to the column based on their affinity for specific molecules or chemical groups.

• The resin contains molecules or ligands that are complementary to specific proteins in the sample.

  • Interact via non-covalent interactions

• The bound protein is released from the resin by passing a solution containing free molecules to compete for binding.

• Useful for concentrating proteins in a small volume

His-tag and Nickel-NTA resin

• Nickle (Ni2+) can coordinate binding of histidine side chains to NTA (nitrilotriacetic acid), but only when the His side chains are close to one another

• A his-tag (6-10 His residues) can be added to proteins to help with purification

  • Strong positive charge helps in binding

• The tagged-protein binds to the column, other proteins will not

• Free imidazole can be used to elute the POI

  • Competing interactions to results in eludating the POI

Immunoprecipitation

• Immunoprecipitation is a form of affinity purification

• Antibodies that specifically bind to a POI or tag are used to trap a POI in a resin

  • The POI contains the antigen

• Protein complexes can be precipitated out of solution (co-immunoprecipitation; Co-IP)

  • Denature all proteins bound to the resin, as well as any interacting proteins (potentially your POI)

• Consideration: can only be used if specific antibodies are available or a tag is added to the POI or “bait”

High Pressure Liquid Chromatography

• Uses very fine beads and high-pressure pumps to move a sample through the column

  • Achieves higher resolution of peaks

• Resin choice determines separation basis, usually silica covered in hydrocarbons

  • Known as Reverse Phase HPLC when separating basedon hydrophobicity

  • In RP-HPLC, hydrophobic compounds move more slowly through the column and have a longer retention time (any compounds that cannot interact will move faster through the column - staying in the mobile phase)

• Issues: only use for smaller proteins

  • Larger proteins may cannot go through at all

Dialysis for protein purification

• Dialysis can be used to remove small molecules (e.g. salts, H+, ions, imidazole)

• Samples are placed in a semi-permeable dialysis bag (i.e. a filter) and incubated with a buffer.

  • Diffusion allows molecules to move from the sample into the buffer.

  • Only for smaller molecules can diffuse out (buffer has less salt - to increase it through diffusion), repeat the step until everything we are not interested in are out of the sample - used until after

• Often used after chromatography

  • Can dilute out (i.e. remove) small molecules used in chromatography that might interfere with other experiments or later assays.

  • Can also be used to change the pH of the buffer (buffer exchange).

Ranking of specifity

Measuring protein

• Biomolecules absorb light at characteristic wavelengths

  • Can be measured using a spectrophotometer

  • Most proteins are colourless and do not absorb visible light (380-750 nm)

  • However, amino acids absorb light in the UV spectrum (100-380 nm)

• The concentration of protein in solution can be measured based on the absorbance at 280 nm, arising from aromatic amino acids (e.g. peptide bonds, aromatic rings)

Protein absorbance

• Stains like Coomassie Blue bind to proteins and absorb light in the visual spectrum

  • Rings can be able to absorb the light effectively and only basic Amino acids

• Can also be used to visualize proteins or quantify the concentration of protein in a Bradford assay

Beer-Lambert Law

• issue with this

• can only determine concentration, but not if your POI is in thtere

SDS-page - separating proteins

• what is SDS + another alternative?

• Sodium Dodecyl Sulfate (SDS) –Polyacrylamide Gel Electrophoresis

  • A amphipathic molecule (some negative and positive polarity regions)

• SDS is a detergent that can denature proteins

  • Disrupt non-covalent interactions

  • 1 molecule of SDS binds approx. every 2 amino acids

  • Gives a polypeptides a uniform (-) charge relative to its mass

• Beta-mercaptoethanol (BME) can also be used to reduce disulfide bonds

• The polyacrylamide gel creates a mesh or sieve of cross-linked molecules that separate subunits based on size

• Under denaturing (SDS) and reducing (BME) conditions polypeptide chains will have the same mass-to-charge ratio and migrate in the gel towards the anode

• Larger proteins will move through the pores slower, allowing for separation of the proteins

• Protein size can be deduced by comparing to MW markers

• Identity needs to be confirmed by immunoblotting or mass. spec.

SDS-page - visualizing proteins

• Now separated in the PAGE gel, the colourless proteins can be visualized through staining

• Protein stains include:

  • Coomassie Blue – binds to basic and hydrophobic amino acids

  • Silver Stains – binds to charged amino acids

  • Fluorescent Dyes

How to determine the purity of your sample?

• need to do multiple purification processes to get the protein you want