Lecture 3 Flashcards
What are the steps to recover a protein from the host cells?
- Extraction
- Enrichment/Isolating the protein of interest
- Purification
- Removal of contaminants
Name the techniques used for cell disruption.
- Homogenizations
- Ultrasonication
- Glass bead milling
- Osmotic shock
- Repeated freezing and thawing
- Enzymatic lysis
- Detergent based lysis
How does homogenization work to disrupt the cell membrane?
The sample is transferred from a chamber at high pressure through a narrow bore orifice into a chamber at low pressure. This rapid pressure drop disrupts the cell membrane.
What are the advantages of homogenization as a cell disruption method?
It is fast and efficient which allows it to be scaled up for large volumes
What are the disadvantages of homogenization as a cell disruption method?
It causes the sample to heat up, which could cause the proteins to denature, so cooling is required.
How does ultrasonication work to disrupt the cell membrane?
The cells are disrupted by high frequency sound waves
What are the advantages of ultrasonication as a cell disruption method?
Very straightforward and simple procedure
What are the disadvantages of ultrasonication as a cell disruption method?
- It causes the sample to heat up, which could cause the proteins to denature, so cooling is required. It can be difficult to control by cooling
- Proteins can get destroyed by shearing due to the sound waves.
- It is noisy so it can’t be used for large volumes
How does glass bead milling work to disrupt the cell membrane?
The cells are agitated with fine glass beads
What are the advantages of glass bead milling as a cell disruption method?
- It is useful for cells that are more difficult to disrupt (e.g. yeast)
- It can be scaled up for large volumes
What are the disadvantages of glass bead milling as a cell disruption method?
It’s relatively slow and noisy
How does osmotic shock work to disrupt the cell membrane?
The cells are transferred from a high to low osmotic medium (15-25% sucrose)
What are the advantages of osmotic shock as a cell disruption method?
Its a simple procedure
What are the disadvantages of osmotic shock as a cell disruption method?
It’s only useful for the disruption of cells with less robust walls like animal cells.
How does freezing and thawing work to disrupt the cell membrane?
The cells get disrupted by repeated formation of ice crystals
This is usually combined
with enzymatic lysis
What are the advantages of repeated freezing and thawing as a cell disruption method?
Its a simple procedure and can yield large membrane fragments, which is good for extracting membrane proteins
What are the disadvantages of repeated freezing and thawing as a cell disruption method?
- Its a slow process with low yield
- The formation of ice crystals may damage sensitive proteins.
What are the advantages of enzymatic lysis as a cell disruption method?
It is a gentle procedure that yields large membrane fragments, which is good for extracting membrane proteins
How does detergent based lysis work to disrupt the cell membrane?
Use detergents like sodium lauryl sulfate to solubilize the cell membrane
How does enzymatic lysis work to disrupt the cell membrane?
Use a lysozyme to disrupt the cell membrane.
This process is often used with other techniques like freezing and thawing and osmotic shock
What are the disadvantages of enzymatic lysis as a cell disruption method?
It’s a slow process with low yield
What are the advantages of detergent based lysis as a cell disruption method?
It’s effective in disrupting the cell membrane, especially in mammalian cells
What are the disadvantages of detergent based lysis as a cell disruption method?
- May induce protein denaturation and precipitation (ionic more likely than nonionic)
- It’s presence may affect subsequent purification steps
- The presence of detergent is unacceptable in final protein preparations –> additional steps have to be taken to remove the detergent
What are the steps done in enrichment?
- Removal of whole cells and cell debris
- Removal of nucleic acid and lipids
- Concentration of protein of interest
- Purification of protein of interest
How does a depth filter work to remove whole cells and cell debris?
- The filter consists of randomly oriented fibers (glass fibers or cellulose) that form a network of mesh-like structures.
- It’s assembled from a series of filters with decreasing pore size rating
- The particles are retained on the surface and within the depth of the filter
How does a membrane filter work to remove whole cells and cell debris?
The filter has a pore size of 0.2-10 micrometers which retains all microbial cells.
How are nucleic acids removed from the product during the enrichment step?
- Precipitation using cationic polyethylenimine
- Treatment with nucleases
How are lipids removed from the product during the enrichment step?
The lipid layer is removed by passing the solution through a glass wool or a cloth of very fine mesh size.
How does salting out help to precipitate the protein?
- Protein solubility varies with ionic strength (I) of solution
- At high I (above an optimal salt conc) → salting out (protein precipitation from solution)
- Salts at high conc compete with proteins for water of hydration → promotes protein-protein interactions between hydrophobic patches on surface of adjacent protein molecules → protein precipitation
How can adjusting the pH of a solution precipitate the proteins out?
The minimum solubility of a protein is generally around its isoelectric point, so altering the pH to its isoelectric point will cause the proteins to precipitate out.
How does the addition of organic solvents help to precipitate proteins from solution?
- The solvation layer around the protein will decrease as the organic solvent progressively displaces water from the protein surface
- This weakens the hydrophobic interactions and disrupts the hydration shell,
- It also increases the electrostatic attractions between proteins of opposite charges, resulting in protein precipitation
What are commonly used organic solvents to precipitate proteins?
Ethanol, isopropanol, acetone
What are commonly used salts to precipitate proteins?
Ammonium sulfate because of it has a high solubility, its cheap and lacks denaturing properties
How does ion exchange work to concentrate a protein solution?
- The solution is passed through ion exchangers.
- For negatively-charged proteins of interest, use Anion exchangers (contain aminoethyl- moieties). For positively-charged proteins of interest, use Cationic exchangers (e.g. Contain carboxymethyl moiety).
- The proteins get bound to the ion exchangers, while undesirable impurities are washed out
- Proteins bound to ion exchangers can be eluted from the ion exchange column by addition of a suitable salt solution of high ionic strength (eg NaCl 0.5 M)
How does ultrafiltration work to concentrate the protein solution?
The solution is passed through a membrane filter (pore size varies depending on protein size)
How does gel filtration chromatography work?
- Gel filtration chromatography separates proteins based on size and shape.
- Proteins are eluted from the column in order of decreasing molecular size
- Proteins that are small enough to enter gel matrix are retained longer on column.
- Proteins that are too large to enter the porous space among gel beads are eluted faster.
How does ion exchange chromatography work?
- It used for charged proteins
- For positively charged proteins, a negatively charged matrix is used and vice versa
- Elution of adsorbed protein is achieved using salt (NaCl) containing buffer.
How does affinity chromatography generally work?
- Ligands are covalently attached to an inert support matrix and packed into a column
- The protein of interest selectively binds to the immobilized ligand and the impurities are eluted out
- The protein of interest is then eluted using different methods
What are the two different approaches for affininty chromatography?
- General ligand approach
- Specific ligand approach
What is the general ligand approach for affinity chromatography?
- Immobilize ATP or cofactors like NAD+ on the affinity column to purify proteins that bind to ATP or cofactors
- The protein of interest is eluted by changing the pH, ionic strength or the polarity of the buffer
- The protein of interest can also be eluted by adding a competing ligand to the eluting buffer so the retained proteins can desorb.
What is the specific ligand approach for affinity chromatography?
- Antibodies complementary to the protein of interest are immobilized onto the affinity column
- The elution of the protein of interest is achieved using denaturing agents like an acidic pH solution (this causes partial denaturation of the protein)
What is done to ensure sterilization of the protein product?
- The biopharmaceuticals are assembled by aseptic procedures which minimizes the amount of microbial contamination
- The protein solution is filtered through 0.2 or 0.22 micrometer membrane filters
What is done in the process of viral decontamination of the protein solution?
Either
1. By heat, gamma irradiation, sonication or extreme pH (harmful to protein)
2. By nanofiltration
How are pyrogens removed from the protein solution?
- Pyrogens are negatively charged endotoxins
- They are removed by dry heat or anion exchange
How may temperature affect protein instability?
high temp promotes protein unfolding by disrupting non-covalent
forces that stabilize protein’s conformation.
denatured proteins aggregate → irreversible denaturation.
How may pH affect protein instability?
- Proteins unfold at extreme pHs due to changes in ionisation status of side chains of aa residues.
- Disruption of distribution of ionic attractive and repulsive forces that stabilise protein’s conformation.
- Extreme pHs also affect protein’s chemical stability → Hydrolysis of Asp residues, and Deamidation of Asn and Gln
How may pH affect protein instability?
- Protein adsorption can cause a significant change in secondary structure and tertiary structure.
- It may also lead to loss of proteins or destabilization of proteins.
How may shaking and shearing affect protein instability?
- Agitation (shaking and shearing) incorporates air into protein solution, creating an air/liquid interface.
- The alignment of proteins along such interfaces can cause unfolding of the protein to maximise the exposure of hydrophobic residues to the air which results in partial or complete protein denaturation.
- Shearing (breaking) also exposes hydrophobic areas.
How may the presence of non-aqueous solvents affect protein instability?
- Protein hydration shells may be disrupted in the presence of a significant amount of non-aqueous solvents.
- Hydrophobic core exposed as polarity of aqueous solvent decreases → Protein unfolds
How may exposure of light affect protein instability?
- Photodegradation
- Risk of protein aggregation upon exposure to light
- Light can cause the side chain cleavage of tryptophan
What are the various factors that can cause physical aggregation of proteins?
- Temperature
- pH
- Adsorption
- Shaking and shearing
- Non aqueous solvents
- Repeated freeze and thaw
- Photodegradation
What are the chemical reactions that may degrade proteins?
- Deamidation
- Oxidation
- Disulfide bond breakage and formation
- Hydrolysis
How does polyethylenimine work to remove nucleic acids?
It binds to the negatively charged DNA and RNA, forming precipitates that can be removed by centrifugation.
