MCB 102 Lec 5 Protein purification

Gotta obtain it

• Biological, natural source
• Bacteria

• Isolate them
• Sequence them
• Look at their structure
• Investigate their chemical properties
• See how they interact with each other

60%

The constellation of proteins in a cell

Purification

When a complex mixture is resolved or disconvoluted into its constituents by the successive application of physiochemical methods for separation and fractionation

• Analysis of its AA sequence
• Examinaton of its functional characteristics
• Determination of its 3D structure

• Mass
• Shape
• Polarity/solubility
• Charge
• Density
• Hydrophobicity
• Ligand-binding specificity and affinity
• Special features

The ratio of the amount of the desired protein present to the amount of total protein present in the same sample

Via its activity in our specific assay

• Assays to detect the presence of the protein of interest at each purification step
• Assays to measure TOTAL protein at each purification step
• Methods to purify the protein

• Colorimetric assays
• Radiochemical assays
• Spectrophometric assay
• Immunochemical assay
• Biological activity assay

• Lowry method
• Bradford method

• A qualitative or quantitative method for detecting the presence and measuring the amt of a biological substance in a complex mixture of other biomolecules
• The action of an enzyme leaves a "molecular footprint"

Molecular footprint

• 1. Either the substrate or product (or both) of the reaction have a specific light absorbance that changes as the reaction progresses
• 2. There is a way to stain for substrate or product after the reaction

• 1. The product of the enzymatic reaction is radioactively labeled
• 2. A by-product of the enzymatic reaction is radioactively labeled

• The absorbance of a product or substrate changes as the reaction progresses
• The whole reaction takes place in a spectrophotometer

Antibodies

• Trying to detect the activity of the enzyme β-galactosidase (cleaving enzyme)
• Galactose is attached to a substance to make the colorless substrate galactoside (X-gal)
• When the substrate is cleaved, it turns blue
• Shows that β-galactosidase was there to cleave
• Concentration of product is assayed by absorbance at 570 nm
• Want excess substrate so amt's of product and concentration of enzyme are linear
• Map enzyme concentration vs. absorbance on a standard curve

Used as a reference

• PI-Scel endonuclease (cuts DNA?)
• Stain DNA with dye that absorbs UV light and emits visible red light

• Hexokinase (glucose phosphorylation; transfers phosphorous group from ATP to RADIOACTIVE glucose)
• Now glucose has a NEGATIVE charge
• DEAE positively charged cellulose filter paper
• ATP, ADP, and P-glucose will bind to filter; glucose will pass
• Find radioactive group stuck to filter using scintillation counter
• Quantify P-glucose
• Compare and map to standard curve

• Theymidylate synthase (enzyme)
• Creates water through condensation
• Receives radioactive/heavy proton from substrate
• Filter through activated charcoal
• All organic molecules will bind, but radioactively labeled water will pass
• Measure activity of enzyme by assessing amt of radioactive water that filtered through charcoal

• Lactate dehydrogenase (reduces NAD⁺ and produces NADH)
• Look for changes in absorbance from light passing through substances
• Because enzyme uses NADH as a substrate, its concentration will decrease according to enzyme concentration

• HIV coat proteins in human serum
• a. Standard enzyme-linked immunosorbant assay (ELISA)
• Antigen → Add specific antibody → Add enzyme-linked antibody → add substrate, get yellow
• Only get reaction if enzyme present; Enzyme only present if antibody binds
• b. Sandwich ELISA
• First antibodies → Add antigen → Add second enzyme-linked antibody → Add substrate, get yellow

• p-nitrophenyl-phosphate (colorless substrate)
• Alkaline phosphatase (antibody coupled enzyme)
• p-nitrophenol (bright yellow product)
• Can also be seen as colorimetric

• Lowry Method
• Bradford method

• Named for the biochemist Oliver H Lowry
• His 1951 paper describing the technique is most-highly cited
• Better if working with membrane potentials
• Not good in cytosol b/c sensitive to the reducing agents normally found there

• Developed by Marion M Bradford (1976)
• Requires very low concentrations of protein (thus samples usually have to be diluted)
• Sensitive to detergents
• Better for cytosolic proteins

• Chemical test for detecting the presence of peptide bonds
• In the presence of peptides, a copper2⁺ ion forms violet-colored coordination complexes in an alkaline soln

B/c peptide bonds occur with the same frequency per amino acid in the peptide

Protein concentration

directly

The intensity of color

• The Biuret test (the rxn of Cu2⁺ ions with the peptides under alkaline conditions)
• The oxidation of aromatic protein residues using the Folin-Ciocalteu reagent

Increases it 100 times

Lowry method

Proteins with basic and aromatic side chains

A standard curve

A buffer

• Aqueous mixture of components that:
• -- extract the desired protein into solution
• -- helps preserve the structure and function of the desired protein

• It resists any change in pH
• It contains other reagents that help prevent the desired protein from suffering unwanted oxidative and/or proteolytic damage

It contains equimolar amounts of a conjugate acid-base pair that has a pKa value at or very near phisiological pH

• Buffering species
• Salt
• Reducing agent
• Chelator
• Protease inhibitor
• Other

To hold pH constant

• Proteins are more soluble at moderate ionic strength
• Helps maintain dielectric constant

To prevent oxidation of Cysteine

To remove heavy metal ions

Substance bonded to a metal ion at two or more points

To revent proteolysis

Extraneous proteins and other contaminants

Specific activity

As purification of protein proceeds till the end

When specific activity remains constant and cannot be further increased

Differential centrifugation

To isolate proteins from specific subcellular fractions/organelles

• Crush/blend tissue completely
• Centrifuge at low speed and separate supernatant
• Repeat but centrifuge at higher speed than before
• Repeat until supernatant is relatively clear
• Supernatant contains soluble proteins
• What steps you take depends on your protein

• Break open the cell
• If the protein is in the cytosol, the crude lysate may be filtered or centrifuged to remove the cell debris and stuff; Protein of interest is in the supernatant
• If the protein is in a membrane or organelle, repeated centrifugation and isolation of pellet or supernatant will provide access to the organelle or membrane

Place cell in hypotonic/osmotic solution

• They have cell walls
• Blender
• Sonicator (ultrasound)
• French press

• After cells have been broken, the crude lysate may be filtered or centrifuged to remove the cell debris
• The protein of interest is in the supernatant

Repeated centrifugation and isolation of the pellet or supernatant will provide access to the organelle or membrane

• We need to know something about the protein
• Some proteins will precipitate if we add salt (usually ammonium sulfate) → "Salting out"
• If we know the approximate size of the protein, we can use a dialysis process with a membrane of the right size
• Column chromatography can separate proteins according to charge, size, affinity for a specific ligand, etc.

• After proteins solubilized, they can be purified based on solubility
• Use ammonium sulfate (NH₄SO₄) as salt
• Takes away water by interacting with it
• Makes protein less soluble b/c hydrophobic interactions among proteins increases
• Aggregation of proteins by their most hydrophobic surfaces b/c no tenough free H₂O for solvation
• Different aliquots taken as function of salt concentration to get closer to desired protein sample of interest (30, 40, 50, 75% increments)
• One fraction has the protein of interest

• Start with a sample of our protein in soluble form
• Run it through a column
• Collect fractons at different times
• Use a biochemical assay to identify the fraction containing our protein

• Stationary
• Mobile

• Solid porous matrix
• Protein samples go through and interact with this phase

• Protein sample
• Flows over the stationary phase
• Carries along with it the sample to be separated

• Start with a sample of our protein in soluble form
• Use a buffer/soln to "push" our protein (mobile phase running down column)
• Proteins will be separated as they will run at different speeds depending on their properties
• Collect fractions of eluate at different times

What you collect during column chromatography

• Gel filtration
• Proteins are separated by size

• Column contains a cross-linked polymer
• Polymer contains small pores
• Proteins that are small enough to enter the pores will be stuck and delayed
• Larger molecules pass more freely; apprearing on the earlier fractions

Separates protein according to charge

• Column contains cation or anion exchangers (polymers w/ charged functional groups)
• Proteins move through the column at rates determined by their net charge at the pH used (and elution buffer used)
• With cation exchangers, proteins with a more negative net charge move faster and elute earlier as they are repelled

Based on ligand binding affinity

• Beads have a ligand covalently attached that will bind the protein of interest and retard its migration through the matrix
• Unwanted species will wash through the column

• After unwanted proteins have washed
• A solution containing the same ligand is now added to the column (could also be high salt or different pH to disrupt interactions)
• The protein is eluted by the ligand solution

Remove by washing withsolvent

• Relatively new technique
• Takes advantage of the fact that most proteins have hydrophobic pockets and of the hydrophobic effect
• Matrix of the column consists of hydrophobic ligands
• Protein solution is mixed with high levels of salt, promoting hydrophobic effect (but not too high b/c can cause ppt)
• Proteins will elute according to their hydrophobicity, with the most hydrophobic proteins beind delayed the most
• Elution is done with solutions of DECREASING salt concentration

Using high performance liquid chromatography

• HPLC
• Uses high pressure pumps that speed up mvmt of the protein molc down the column
• Speical columns that can sustain the pressure of the pressurized flow (no compressible resin and strong columns)
• Results in reduced transit time which limits the diffusion of the protein and enhances the resolution

• Reduced transit time
• Limits the diffusion of protein
• Enhances resolution

For protein analysis

By electrophoresis

• Electric field pulling on proteins according to their charge
• Gel matrix hinders mobility of proteins according to their size and shape

• Allows us to visually separate proteins and assess the degree of purity of a mixture
• Can also determine the MW and pI of proteins

• Different samples are loaded in wells or depressions at the top of the polyacrylamide gel
• The proteins move into the gel when an electric field is applied
• The gel minimizes protein mvmts other than those induced by the electric field

Their size and shape

• In their native form
• Bound to sodium dodecyl sulfate (SDS)

• Denatures the protein (no 3D structure interference)
• Makes the protein negatively charged with overall charge being proportional to the length of the protein
• Rate of mvmt will only depend on size, with smaller polypeptides migrating more rapidly

Proteins bind approximately 1 molecule of SDS/AA

Gel Loading Buffer

• It has glycerol (density)
• It has bromophenol blue (dye)

• Density
• Need the protein to stay at bottom of well/groove until it's time for electricity

• It's a dye that can be seen by the naked eye
• Used to monitor the migration of proteins in the gel

Has proteins of different known sizes

Used to estimate the size of the protein

• Once the gel has run, it's placed in a Comassie Blue soln that binds to proteins
• Each band in the gel represents a different protein or protein subunit
• Can see different purification steps

The molecular weight

A procedure used to determine the isoelectric point of a protein

• A pH gradient is established in a gel
• When a protein mixture is applied, each protein migrates until it reaches the pH that matches its pI

• Isoelectric focusing and SDS-PAGE are combined
• Proteins are first separated by isoelectric focusing in a thin strip gel
• The gel is tehn laid horizonally on a second, slab-shaped gel (SDS-PAGE)
• The proteins are separated by SDS polyacrylamide gel electrophoresis
• Thus, the original protein mixture is spread in two dimensions

Reflects differences in pI

Reflects differences in molecular weight

• Protein pools
• (treatment, diseases, etc.)