Chapter 2 Introduction to protein purification Flashcards
Objectives
Describe the process of cell breakage and protein extraction.
Describe the general principles, strategies and techniques involved in the purification of proteins.
Describe methods used in the assay of proteins.
Under sample preparation between the workflow of sample and protein mixture
Sample separation and visualization
Comparative analysis
Digestion
Why does a sample need to be prepared
- solubilize proteins from source into a stable, liquid form for subsequent procedures
- remove contaminants or unwanted proteins that might affect resolution during visualization e.g (SDS-PAGE)
Cell Disruption
Depends on the type of materials from which to extract proteins
3 types of cell disruption
Serum, plasma, urine = liquid samples, can be used directly with little pre-treatment
Cells = need for lysis
Tissues = need for homogenisation through mechanical means before lysis
(e.g. plant tissues, fibrous tumours etc.)
Methods of physical lysis / disruption
- Sonication
Cell suspensions samples subjected to sonication in short bursts to avoid heating and foaming.
High frequency sound waves generated by sonication creates shear forces that lyse cells.
methods of physical lysis / manual grinding
- Manual grinding
Grinding by mortar and pestle- tissue or cells often frozen in liquid nitrogen and ground to fine powder
methods of physical lysis / mechanical disruption
- Mechanical disruption
Hand held devices e.g. Dounce homogenizer or blenders can be used to disrupt cell suspensions
5 methods of physical lysis
sonication, manual grinding, mechanical disruption, liquid homogenization, rapid freeze thawing
methods of physical lysis / liquid homogenization
Cells are lysed by shear forces resulting from forcing the cells suspension through
a small opening under high pressure in a French pressure cell.
methods of physical lysis / rapid freeze thawing
Freezing creates ice crystal formation that disrupts cell membranes and if followed by rapid thawing, the cells burst.
2 methods of enzyme or detergent based lysis
Enzymatic lysis and detergent lysis
enzymatic lysis
Used to digest cell walls of microbes and plants
e.g.
cellulase for plants, 1,3-glucanase for yeast and lysozyme for Gram-negative bacteria.
detergent lysis
Uses detergents to disrupt cell membranes
Ionic detergents e.g. SDS (-ve) and CTAB (+ve)
Zwitterionic detergent e.g. CHAPS
Non-ionic detergent e.g. NP-40
Typical lysis protocol for mammalian cells
- Harvest 5×106 cells and wash with PBS
- Add 200µl of lysis buffer
- Spin at 14,500 rpm for 1 hr at 4ºC
- Store at -80ºC
Composition of typical lysis buffer
Urea (7M) Thiourea (2M) CHAPS (4%w/v) TRIS Base (40mM in water) DNAse (10mg/ml) RNAse (10mg/ml) PMSF (0.1M) DTT (1 mM)
chaotropic agents in typical lysis buffer
Urea (7M)
Thiourea (2M)
CHAPS (4% w/v)
Detergent
TRIS-base (40mM in water)
Buffer
DNAse (10mg/ml)
Digest DNA/RNA
RNAse (10mg/ml)
Digest DNA/RNA
PMSF (0.1M)
Protease inhibitor
DTT (1mM)
Reductant
chaotropic agents
Cosolutes that can disrupt the hydrogen bonding network between water molecules and reduce the stability of the native state of proteins by weakening the hydrophobic effect.
The 3 methods of detergent lysis
- Solubilising membrane proteins and lipids.
- Controlling protein crystallization.
- Preventing non-specific binding in affinity purification
and immunoassay procedures
Additional function of detergent
Detergents also promote electrophoresis of soluble proteins (e.g. SDS-PAGE)
Additional function of detergent
Able to disperse hydrophobic proteins or hydrophobic parts of proteins.
Detergent/surfactant molecules have both hydrophilic and hydrophobic portions
Detergents for lysis
CHAPS (zwitterionic), NP-40 (non-ionic) or Triton-X (non-ionic) are commonly used (compatible with IEF)
SDS (anionic) may be used sparingly for difficult samples
Types of detergent to be used
Detergents used must be zwitterionic or nonionic to prevent complications during downstream applications e.g. isoelectric focusing (IEF).
Solubilizing action of detergents
before solubilization
after solubilization
Structure of detergents (CHAPS)
CHAPS (zwitterion)
There is a hydrophobic and hydrophilic halves of the molecules
Structure of detergents (NP40)
NP40 (monionic)
There is a hydrophobic and hydrophilic halves of the molecules
Hydrophobic is C8H17 bound to a benzene
Solubilizing effect of detergents
Proteins are held in the lipid bilayer by hydrophobic interactions between the lipid tails and hydrophobic protein domains.
- These integral membrane proteins (IMPs) are not soluble in aqueous solutions as they aggregate to protect their hydrophobic domains, but are soluble in detergent solutions as micelles formed by detergents are analogous to the bilayers of the biological membranes.
- Proteins are incorporated into these micelles via hydrophobic interactions. Hydrophobic regions of membrane proteins, normally embedded in the membrane lipid bilayer, are now surrounded by a layer of detergent molecules and the hydrophilic regions are exposed to the aqueous medium.
This keeps the membrane proteins in solution.
Complete removal of detergent could result in aggregation due to the clustering of hydrophobic regions and, hence, may cause precipitation of membrane proteins
Micelle
The inside is hydrophobic and outside is hydrophillic
Native membrane -> solubilization and purification
Chaotrope or chaotropic agents in slides
A chaotrope disrupts hydrogen bonding and hydrophobic interactions between and within proteins.
Result of addition of chaotropic agents
This action breaks proteins and convert proteins from native conformation into a random conformation thereby solubilising them
Why urea is used at 8M
Urea used at 8M unfolds most proteins
Why urea is used with thiourea
Urea is used together with thiourea to solubilise membrane proteins.
Thiourea increases solubilisation power of urea.
Urea/thiourea has to be prepared fresh to reduce conversion to cyanate/thiocyanate.
Cyanate/thiocyanate can cause carbamylation which changes MW of solubilized proteins.
Carbamylation
Carbamylation is a non-enzymatic spontaneous reaction of a primary amine or a free sulfhydryl group of protein with isocyanate
Carbmylation as a problem
In 2-D gel analysis and the associated sample preparation steps.
This modification occurs when iso-cyanate, a urea break-down product, covalently modifies lysine residues, thus inducing a change in isoelectric point.
It also changes the MW of solubilized proteins
How do carbamides form
Isocyanic acid reacts withaminesto giveureas(carbamides)
Carbamylation
HNCO + RNH2→ RNHC(O)NH2
Reductants or reducing agents
Reducing agents or reductants break apart intramolecular and intermolecular disulphide bonds in proteins and maintain them in fully reduced state (-SH)
DTT (dithiothreitol)
Commonly used as a reducing agent
Reason DTT is preferred over beta-mercaptoethanol
DTT is preferred over β-mercaptoethanol because of its water solubility and lower toxicity.
Danger with beta-mercaptoethanol
2‑Mercaptoethanol is considered toxic, causing irritation to the nasal passageways and respiratory tract upon inhalation, irritation to the skin, vomiting and stomach pain through ingestion, and potentially death if severe exposure occurs.
Why do we use detergents, chaotropes and reductants
Various components of the lysis buffer break apart proteins to denatured forms.
Protein-protein interactions complicate identification.
There is a need to reduce proteins to their smallest unit possible.
Small issue with 2D-PAGE
Note, some protein complexes are too large to be resolved using 2D-PAGE.
Removal of contaminants : DNA/mRNA
Reasons DNA/mRNA is removed
How DNA/mRNA is removed
DNA and mRNA are released into lysis buffer.
DNA/mRNA in prepared sample increases viscosity of the soluble protein and makes handling difficult (e.g. pipetting).
Some protein form complexes with DNA/mRNA.
We add nucleases (DNase/Rnase) digests DNA/mRNA
Removal of contaminants : cell debris
How cellular membrane fragments are moved
cellular membranes remain as fragments after lysis
We remove them with simple step of centrifugation
- cellular membrane fragments will pellet after centrifugation while soluble proteins remains in the supernatant
Typical lysis protocol for mammalian cells
Spin at 14.500 rpm for 1 hour at 4 degree Celsius
Temperature for storage and handling of samples
Store at -80 degree Celsius
Typical lysis protocol for mammalian cells
- Harvest 5×106 cells and wash with PBS
- Add 200µl of lysis buffer
- Spin at 14,500 rpm for 1 hr at 4ºC
- Store at -80ºC
Proteases released during cell lysis
Proteases are released by cells during lysis, when the lysosomes of cells are compromised
Proteases degrade proteins and cause problems during sample preparation
3 methods of handling proteases released during cell lysis
Prepare and handle sample at low temperature (e.g. on ice).
Long-term storage at -80ºC or -20ºC.
Add protease inhibitors to preserve quality of proteins used for subsequent steps.
Endopeptidases (common proteases)
A broad range of enzymes catalyze the hydrolysis of peptide bonds in the interior of a polypeptide chain or protein molecule
Mechanism of action by endopeptidases
Nucleophilic attack by OH-group on the carbonyl group of the peptide bond
Thus, enabling the cleavage of the peptide bond via hydrolysis
5 common protein inhibitors
PMSF EGTA/EDTA Leupeptin Pepstatin Aprotinin
PMSF
Against serine, cysteine proteases
EGTA/EDTA
Against metalloproteases
Leupeptin
Against serine/cysteine proteases
Pepstatin
Against aspartyl proteases
Aprotinin
Against serine proteases
Example of sample preparation protocol by proteomic analysis
- Protease inhibitors
- Freeze thawing
- Detergent and reductant
- Nucleases
- Protease inhibitors, detergent
- Chaotropic agents detergent
Protein prefractionation
Prefractionation may be carried out to enrich for specific proteins
> Studying proteins associated with certain cellular organelles/compartments or studying certain classes of proteins
> Some proteins occur at a low abundance relative to other
Plasma/serum immunoglobulins are abundant and thus need to remove abundant unwanted proteins
2 reasons for prefractionation
- Reducing the amount of unwanted proteins increases the amount of proteins that can be loaded on 2D-PAGE.
This facilitates detection of low abundance proteins.
- The loading of protein on an IEF gel is limited at a maximum ~120µg)
2. Reducing protein complexity improves resolution in 2D-PAGE.
Use of ion exchange chromatography for prefractionation
Separating a sample mixture by applying it through a medium in which different components move at different rates
Pre fractionation
Prior fractionation of proteins
Try to use a fractionation methods that generates minimal protein overlap between fractions
3 common methods of prefractionation
- immunoaffinity depletion
- affinity chromatography
- subcellular fractionation
Principles of Immunoaffinity depletion
A form of affinity chromatography.
Antibodies to “unwanted” proteins are immobilized onto a solid matrix in a column.
Protein samples are applied to the column and “unwanted” proteins bind to the column.
Remaining “wanted” proteins flow through and are collected.
Immunoaffinity depletion
“Unwanted” proteins trapped and remaining :wanted proteins flow through and collected
Affinity chromatography
“Wanted proteins trapped and eluted later”
“Unwanted proteins flow through and discarded”
Antibody affinity chromatography
1. Load in pH7 buffer Proteins recognized by antibodies Protein not recognized by antibodies 2. Wash 3. Elute with pH 3 buffer
Subcellular fractionation
To enrich the sample with certain organelles (nuclear or cytosolic fractions, ER, mitochondria) for investigation of certain classes of proteins.
Mechanical homogenisation followed by various steps of centrifugation and/or ultracentrifugation.
Process of subcellular fractionation
Filter homogenate to remove clumps of unbroken cells, connective tissues etc
Filtered homogenate 600g x 10 min
Nuclei pour out 15,000g x 5 min
Mitochondria, chloroplasts, lysosome and peroxisome pour out 100,000g x 60 minutes
Plasma membrane, microsomal fraction (fragments of endoplasmic reticulum) and large polyribosomes
pour out 300,000g x 2 hours
Ribosomal subunits, small polyribosomes pour out soluble portion of cytoplasm
Ultra centrifugation vs centrifugation
Using different centrifugal forces allow fractionation of cellular components of various densities
Ultracentrifugation is 80,000g while centrifugation is 800g
Why use a mix of ultra centrifugation and centrifugation
Using different centrifugal forces allow fractionation of cellular components of various densities
5 examples protein quantification and detection methods
UV spectrometry
Colorimetric assays
Other methods: (e.g Amino acid analysis, radio-labelling, RP-chromatography)
principle of UV-spectrophotometry
Absorbance of light at near UV (280 nm) by protein is dependent mainly on tyrosine and tryptophan content and to a lesser extent phenylalanine
Advantages of protein determination using UV-Vi spectrophotometry
- Method is simple and fast.
- The sample can be recovered.
Disadvantages of protein determination using UV-Vi spectrophotometry
Interference from other chromophores.
Specific absorption value for a given protein must be determined.
Presence of contaminant nucleic acid significantly increases absorbance at 280 nm.
Examples of principle of protein denaturation using UV-spectrophotometry
Cuvette-based spectrophotometer, Nanodrop device
Colorimetric assays for protein quantification
Quantification of total protein content is required to determine sample loading
Commonly employed assays include
- Bicinchoninic acid assay (BCA)
2. Bradford assay
BCA(bicinchoninic acid) assay 2 steps
Step 1: Biuret reaction
Protein + Cu+2 -> Cu+1 Reduction fvia OH-
Step 2:
Cu-1 + 2BCA+ -> BCA+ Cu complexe
Principles of the 2 steps in bicinchoninic acid
Step 1: The BCA assay method involves the reduction of cupric ions to cuprous ions by proteins, in an alkaline reaction (biuret reaction)
Step 2: 2 molecules of bicinchoninic acid chelates with 1 molecule of cuprous ion to form a purple colored complex that absorbs light at 562nm
Note: purple complex is read at 562nm on spectrophotometer
Cupric
Copper with a valency of two; of copper(II).
Cuprous
Copper with a valence of one.
Bradford assay
Protein (basic and aromatic side chains) + Coomassie* G-250 forms a protein dye complex
is blue at 595nm
Principles of bradford assay
Proteins containing basic or aromatic amino acid side chains bind by hydrophobic or Van der Waal’s interactions with Coomassie Brilliant Blue dye.
This causes a spectral shift from red/brown form of the dye (absorbance at 465 nm) to the blue form of the dye (absorbance at 595nm)
The more blue the bradford assay, the more proteins it has
Recap of basic and aromatic amino acids
Basic side chains - lysine, histidine, arginine
Aromatic side chains - tyrosine, phenylalanine and tryptophan
Substances that interfere with protein colourmetric assay
Certain substances react directly with reagents in protein assay, giving “higher” inaccurate readings
Different thresholds for different assays
Choice of assay depends on the composition of sample
What is threshold in protein colorimetric assay?
Threshold is the amount of the interfering substance if exceeded will interfere with assay
Tolerances for detergents and reducing and thiol containing agents of BCA by concentration
Brij - 35 5.0%
Brij - 56 1.0%
Brij - 58 1.0%
N-acetylglucosamine in PBS pH7.2 10mM
Ascorbic acid –
Cysteine –
Tolerances for detergents and reducing and thiol containing agents of BCA by Coomassie assay
Brij - 35 - 0.125%
Brij - 56 - 0.031%
Brij - 58 - 0.031%
N-acetylglucosamine in PBS pH7.2 100mM
Ascorbic acid 50 mM
Cysteine 10 mM
Comparison of assays (BCA part)
Less variability than bradford
Slower than bradford as it has more steps
Compatible with detergents such as SDS and triton-X
Better detection range (20-2000ug/ml)
Comparison of assays (Bradford part)
More variability than BCA
Faster than BCA
Compatible with reducing agents
Detection range (1-1,400ug/ml)
Know what is the problem with a protocol
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